Regulations Of Venous Molecular Marker COUP-TF? On Thesenescence Of Vascular Endothelial Cells

Background:Atherosclerosis(AS),one of the main factors leading to death and disability,seriously impacts the health and quality of lives in modern society.Atherosclerosis is the major pathological cause of various vascular diseases.Veins and arteries present in the same vascular system.However,atherosclerosis occurs in arteries but not in veins.Regarding the different susceptibility of arteries and veins to atherosclerosis,ample evidences regarding hemodynamics had been reported.With the rapid development of embryo vessels,researchers realized that arteries and veins owned different molecular phenotype.However,it remains largely unknown whether the different susceptibility of arteries and veins to atherosclerosis is related to their molecular phenotype.Arteries and veins have been reported to present different molecular phenotype during embryo development.As one member of the orphan nuclear receptor superfamily,chicken ovalbumin upstream promoter-transcription factor II(COUP-TF?)plays the key role in the differentiation of veins and arteries.It has been reported that COUP-TF? maintains venous identity by suppressing NP-1,Jagged1 and Notch1 signaling expression in the vascular system.Besides the regulations on the differentiation of embryo vessels,COUP-TF? exerts multiple pathophysiological regulations including oxidative metabolism,oxidative stress and renin-angiotensin system,which are the important pathophysiological processes leading to occurrence of atherosclerosis.Therefore,it suggested that COUP-TF? gene,the venous phenotype-specific molecular marker,might play an important role in the regulation of the susceptibility to atherosclerosis.Endothelial cells(ECs)play important roles in the maintaining of normal function of vessels,and the senescence of ECs is the important intiation factor for AS.Based on previous studies,we hypothesized that COUPTF? might influence the susceptibility of vessels to AS by regulating on the senescence of vascular ECs.Objective:We studied the effects of COUP-TF? on the senescence and proliferation of vascular endothelial cells as well as its possible signal and molecular mechanisms.Methods:1.Human umbilical vein endothelial cells(HUVECs)and human coronary artery endothelial cells(HCAECs)were cultured and the m RNA expression of COUP-TF? gene in vitro was detected by PCR.2.The expression of COUP-TF? in HUVEC was knocked down by COUP-TF?siRNA transfection,and the effects of COUP-TF? on the senescence and proliferation of vascular endothelial cells were observed.After stimulating by AngII(10-5 mol/L)for 48 h,the senescence of HUVEC was detected by ?-galactosidase(?-gal)staining,intracellular reactive oxygen species(ROS)production of fluorescence probe DCFH-DA,and P53 expression of western blot.The proliferation of HUVEC was detected by CCK-8 and cell counting.3.To further understand the role of Akt signaling in the regulation of COUP-TF? on ECs senescence,the protein expression of Akt / p-Akt was detected by western blot.After adding the Akt activator SC79(4ug/ml),the senescence rate of HUVEC and the protein expression of senescence-related gene P53 were detected by ?-gal cell staining or intracellular ROS production and western blot.The proliferation of HUVEC was detected by CCK-8 and cell counting.The possible mechanisms of Akt signaling and COUP-TF? on the senescence and proliferation of vascular endothelial cells were observed.4.To further understand the role of Insulin-like growth factor-binding protein 1(IGFBP-1)in the regulation of COUP-TF? on ECs senescence,the protein expression of IGFBP-1 was detected by western blot.After adding the IGFBP-1 protein(100ng/ml),the senescence rate of HUVEC and the protein expression of senescence-related gene P53 were detected by ?-gal cell staining or intracellular ROS production and western blot.The proliferation of HUVEC was detected by CCK-8 and cell counting.The possible mechanisms of IGFBP-1 and COUP-TF? on the senescence and proliferation of vascular endothelial cells were observed.5.Micro RNA(miRNA)chip analysis was performed to understandthe regulation of COUP-TF? on the mi RNA profile.Without Ang II induction,three RNA samples of control group and si-COUP-TF? group were extracted respectively in order to mi RNA One Array? chip analysis by BGI Tech Company.Results:1.Compared with the control groupknocking-down the expression of COUP-TF? in HUVEC via siRNA transfection significantly promotedAng II(10-5mol/L)induced senescence of endothelial cell:we respectively detected ?-gal staining positive cell rates,ROS positive cell rates of fluorescence probe DCFH-DA,and P53 protein expression of western blot in theendothelial cell.At AngII 0mol/L,compared with control group,?-gal staining positive cell rates(0.0622±0.0192 vs 0.0665±0.0132;P>0.05),ROS positive cell rates(0.0931±0.0111 vs 0.1120±0.0125;P>0.05)and P53 protein expression(1.0000± 0.0544 vs 0.9870±0.0794;P>0.05)were no significant difference in the si-COUP-TF? group;At AngII 10-5 mol/L,compared with control group,?-gal staining positive rates were increased(0.4390±0.0673 vs 0.8107±0.0452;P < 0.05),ROS positive cell rates were increased(0.1536±0.0145 vs 0.3306±0.0532;P<0.05)and P53 protein expression was increased(1.0000±0.0956 vs 1.4142±0.0633;P<0.05)in the si-COUP-TF? group.2.Compared with the control groupknocking-downthe expression of COUP-TF? in HUVEC via si RNA transfectionsignificantly inhibited the AngII(10-5mol/L)induced proliferation of endothelial cell:we respectively detected relative OD value at 450 nm and cell counting in the endothelial cell.At AngII 0mol/L,compared with control group,relative OD value(1.0000±0.0272 vs 0.9839±0.0315;P>0.05)and cell counting((1.0417± 0.0892)?105 vs(1.0938±0.0970)?105,P>0.05)were no significant difference in the si-COUP-TF? group;At Ang II 10-5 mol/L,compared with control group,relative OD value was decreased(1.0000±0.0808 vs 0.5743±0.0567;P<0.05)and cell counting was decreased((3.5188±0.0963)?105 vs(2.0875±0.0973)?105;P<0.05)in the si-COUP-TF? group.3.Akt activator SC79(4ug/ml)can partially reverse the regulations of COUPTF? on the senescence and proliferation of endothelial cell: we respectively detected ?-gal staining positive cell rates,ROS positive cell rates of fluorescence probe DCFH-DA,and P53 protein expression of western blot in theendothelial cell.After adding Akt activator SC79 with AngII 10-5 mol/L treatment,?-gal staining positive cell rates were decreased(0.4436±0.0488 vs 0.7558±0.0667 vs 0.5154±0.0213,P<0.05),ROS positive cell rates were decreased(0.1325±0.0221 vs 0.3404±0.0383 vs 0.2133±0.0205,P<0.05)and P53 protein expression was decreased(1.0000±0.0665 vs 1.4985±0.0710 vs 1.0818±0.0603,P<0.05)in the si-COUP-TF?+SC79 group.We respectively detected relative OD value at 450 nm and cell counting in the endothelial cell.After adding Akt activator SC79 with AngII 10-5 mol/L treatment,relative OD value was increased(1.0000±0.0613 vs 0.5940± 0.0296 vs 0.8250±0.0385,P<0.05)and cell counting was increased((4.3667± 0.0933)?105 vs(1.6417±0.0969)?105 vs(2.6958±0.0966)?105,P<0.05)in the si-COUP-TF?+SC79 group.4.IGFBP-1 protein(100ng/ml)can partially reverse theregulations of COUPTF? onthe senescence and proliferation of endothelial cell: we respectively detected ?-gal staining positive cell rates,ROS positive cell rates of fluorescence probe DCFH-DA,and P53 protein expression of western blot in theendothelial cell.After adding IGFBP-1 protein with Ang II 10-5 mol/L treatment,?-gal staining positive cell rates were decreased(0.2121±0.0412 vs 0.6423±0.0286 vs 0.2616±0.0318,P<0.05),ROS positive cell rates were decreased(0.1382±0.0110 vs 0.3357±0.0242 vs 0.2620±0.0379,P<0.05)and P53 protein expression was decreased(1.0000±0.1018 vs 1.4626±0.0459 vs 1.1167±0.0481,P<0.05)in the si-COUP-TF?+IGFBP-1 group.We respectively detected relative OD value at 450 nm and cell counting in the endothelial cell.After adding IGFBP-1 protein with AngII 10-5 mol/L treatment,relative OD value was increased(1.0000±0.0717 vs 0.6051±0.0626 vs 0.9058±0.0607,P<0.05)and cell counting was increased((4.4667±0.0707)?105 vs(2.1583±0.0905)?105 vs(3.6271±0.0898)?105,P< 0.05)in the si-COUP-TF?+ IGFBP-1 group.5.Knocking-down the expression of COUP-TF? in endothelial cell resulted in the changes of 27 mi RNAs,with 21 mi RNAs up-regulated and 6 miRNAs down-regulated.In those changed mi RNAs,miR-642a-3p was related to the lipid metabolism;miR-513a-5p?miR-130a-3p?miR-221-3p and miR-652 were related to inflammation;mi R-196a?miR-1247?mi R-92 b and miR-363-5p were closely related to cell proliferation.Conclusion:1.Knocking-down the expression of COUP-TF? can promote the senescence and inhibit the proliferation of endothelial cell with AngII stimulation,suggesting a protective effect of COUP-TF? on endothelial cell.The molecular mechanism might be related to the regulation of COUP-TF? on Akt signaling and IGFBP-1 expression.2.Knocking-down theexpression of COUP-TF? can change the mi RNAs expression profile of venous endothelial cellsmainly involving inflammation,lipid metabolism and cell proliferation,suggesting that COUP-TF? might affect the expression profile of mi RNAs and thus affect the atherosclerotic susceptibility of vessels.