Effects Of Oxidized Low Density Lipoprotein And Atorvastatin On Angiotensin-converting Enzyme 2 In Human Vascular Endothelial Cells

BackgroundThe Rcnin Angiotcnsin System(RAS) plays a major role in atherogenesis, Angiotensinâ…¡(Angâ…¡) was the main effector peptide of this system.In recent study,potential interaction between Angâ…¡and oxidized Low Density Lipoprotein (ox-LDL) plays a key role in the initiation and progression of atherosclerosis, Angâ…¡contribute to atherogenesis by increasing oxidation and uptake of Low Density Lipoprotein(LDL) and promoting uptake of ox-LDL through Lectin-like Oxidized Low Density Lipoprotein Receptor-1(LOX-1).On the other hand, ox-LDL can regulate the expression of components of RAS and increase the physiopathologic effect of Angâ…¡.RAS blockade exerts potent antiatherosclerotic effects,which are mediated by their antihypertensive,anti-inflammatory, antiproliferative and oxidative stress-lowering properties.Angiotensin-converting Enzyme 2(ACE2) is a newly described enzyme that can break down angiotensinâ… into Angiotensin(1-9)(Ang(1-9)) and Angâ…¡into angiotensin(1-7) (Ang(1-7)).There are 60-80%formation of the angiotensinâ…¡in extracts from huaman vascular tissues is dependent on chymase,the pathway of Angâ…¡formation is not only affected by Angiotensin-converting Enzyme Inhibition(ACEI).ACE2 can bc atheroprotective because it removes Angâ…¡from the atherogenic milieu. Atorvastatin is the newest 3-hydroxy-3-methylglutaryl-CoA(HMG CoA) reducing enzyme inhibitor.It has been demonstrated that the beneficial effect of statins on cardiovascular events such as anti-atherogenesis is not only attributed to cholesterol-lowering,but also to various effects on the vascular wall,which include improved endothelial function as well as antioxidant and anti-inflammatory activity.Ox-LDL and its receptors have been identified in the atherosclerotic vessel walls.ox-LDL via LOX-1 activation upregulates ACE gene expression in endothelial cell and AT1R gene expression in smooth muscle cell.The present study was designed to examine 1) if ox-LDL regulates the gene and protein expression of ACE2 in human umbilical vein endothelial cells(HUVECs),2) if atorvastatin, a potent HMG CoA reductase inhibitor,upregulates the expression of ACE2 in HUVECs,and lastly 3) if atorvastatin influences the effects of ox-LDL on ACE2 expression.Objective1.To determine the effects of ox-LDL on gene and protein expression of ACE2 in HUVECs.2.To investigate whether atorvastatin could regulate gene and protein expression of ACE2 in HUVECs which was elicited by Ox-LDL.Subjects and Method1.SubjectsThe cultured human umbilical vein endothelial cells.Design of experiment: an observational and controlled experiment.2.Method2.1 Preparation of ox-LDLWe separated and quantitatified the LDL from blood of healthy individuals by Super high density gradient centrifugation,and adjusted it to a final concentration of 500ug/mL,then incubated with 10mmol/mL CuSO4.The oxidation was stopped with 100μmol/L EDTA.Finally,we used the method of Bradford to detect the protein level of ox-LDL.2.2 Preparation of atorvastatinCrude drug of atorvastatin(60.47mg) was dissolved in 1ml pure alcohol, 0.1mol/L NaOH of 1.5ml was added.Then the mixture was heated at 55℃for 2 hours,and PH value was regulated with 0.1mol/L HCl to be 7.2,then PBS was added to 5ml and 10mmol/L reserved atorvastatin solution was prepared and stored at-70℃.2.3 Culture and passage of HUVECsThe freezing HUVEC lines were inoculated in M199 medium supplemented with 50ng/L ECGS,5kU/L heparin,1%Penicillin,100mg/L streptomycin and 10%fetal bovine serum(FBS) after they were thawed.The culture medium was changed every two days and confluens of HUVECs was typically achieved in 5-8 days with "cobblestone appearance" in optical microscopy,then 0.25%trypase were used in passage of HUVECs at re-cultured to subconfluence.Cells of 3～6th passage were initially trypsinized,transferred to 10cm² culture dishes with the concentration of 2.0×10⁶ cells/well and re-cultured for 24h in a humidified 95% air and 5%CO₂ atmosphere at 37℃.then they were cultured with serum-free medium and rolled into the experiment.2.4 groups and cultural condition2.4.1 Effects of ox-LDL on gene and protein expression of ACE2(1) HUVECs cultured with equal serum-free medium as control;(2-4) Different concentration groups of ox-LDL:HUVECs were treated with ox-LDL(20mg/L,40mg/L and 80mg/L) for 24h,respectively;(5-7) In a time-control experiment groups,HUVECs were treated with ox-LDL at final concentration of 40mg/L for 6,12 and 24h,respectively.2.4.2 Atorvastatin and the expression of ACE2 in response to ox-LDL. (1) HUVECs cultured with equal serum-free medium as control;(2-4) Different concentration groups of atorvastatin:HUVECs were treated with atorvastatin(1umol/L,5umol/L and 10umol/L) for 24h,respectively;(5-7) In a time-control experiment groups,HUVECs were treated with atorvastatin at final concentration of 5umol/L for 6,12 and 24h,respectively.(8-10) Coculture groups 8:HUVECs were cultured with 40mg/L ox-LDL for 24h as control.9-10:HUVECs were preincubated with atorvastatin (1,10umol/L,respectively) for 1h and then cocultured with ox-LDL(40mg/L) for 24h.2.5 Semiquantitative RT-PCR for ACE2ACE2 mRNA was examined by RT-PCR.Total RNA extracted from cultured HUVECs was reverse-transcripted using specific primers for human ACE2.The products of PCR amplified samples were visualized on 1.5%agarose gels using ethidium bromide.Each specific mRNA band was normalized with GAPDH mRNA band.2.6 Western Analysis for ACE2HUVECs lysate from each experiment was separated by SDS-PAGE,and transferred to nitrocellulose membranes.After incubation in blocking solution, membranes were incubated with 1:250 dilution primary antibody to human ACE2 for overnight at 4℃.Membranes were washed and then incubated with 1:500 dilution second antibody for 1h,and the membranes were detected with the ECL system, and relative intensities of protein bands were analyzed by Scan-gel-it software.3.Data analysisSPSS 11.5 statistical soft was used.Data shown were from six independently performed experiments.Data are presented as mean±S.D.Statistical significance was determined in multiple comparisons among different groups of data in which one-way ANOVA test indicated the presence of significant differences.P value≤ 0.05 was considered significant.Results1.Identification of HUVECs.The confluens of HUVECs was typically achieved in 5-8 days with "cobblestone appearance" in optical microscopy,the confluent cells show contact inhibition.The positive result from immunostaining of factorâ…§related antigen in cultures was 100%.it supported that the cultured cells was HUVECs,The trypan blue stain was used to test the cell activity,the survival rate of HUVECs was over 96%.2.Effects of ox-LDL on ACE2.Incubation of HUVECs with ox-LDL(20,40 and 80mg/L) for 24h significantly decreased the expression of ACE2(mRNA and protein) in a concentration-dependent manner(F=57.438,P＜0.001å’ŒF=42.299,P＜0.001).When compared with control group,ACE2 mRNA expression decreased to 73%,51%,33%(P＜0.001,respectively) and protein expression decreased to 81%,50%and32%(P=0.010,P P＜0.001,P＜0.001,respectively), after treated with different concentrations of 20,40 and 80mg/L ox-LDL.Incubation of HUVECs with ox-LDL(40mg/L) for 6,12 and 24h markedly decreased the expression of ACE2(mRNA and protein) with control group (F=36.483,P＜0.001 and F=24.985,P＜0.001).When treated with ox-LDL for 6,12 and 24h at concentration of 40mg/L ox-LDL,ACE2 mRNA expression in HUVECs decreased to 66%,56%and 50%when compared with control group(P＜0.001,respectively),and protein expression decreased to 63%,53%and 49% (P＜0.001,respectively).3.Relationship between atorvastatin and the expression of ACE2Gene expression and protein expression of ACE2 in groups treated with atorvastatin alone showed no significant difference in both concentration and time dependent manners when compared with that of control groups(P＞0.05).4.Role of atorvastatin in the expression of ACE2 excited by ox-LDL.Preincubation of HUVECs with atorvastatin significantly inhibited ox-LDL-induced mRNA and protein expression of ACE2 in response to ox-LDL (F=28.199,P＜0.001 and F=20.321,P＜0.001).Compared with 40mg/L ox-LDL alone group,group which pretreatment of HUVECs with atorvastatin(10 umol/L) for 1h and then coculture with 40mg/L ox-LDL for 24h stimulated 1.75 fold increase in the ACE2 gene expression and 1.69 fold increase in the ACE2 protein expression(P＜0.001,respectively).Group which coculture with 1umol/L atorvastatin and 40mg/L ox-LDL differed no significantly to group without atorvastatin pretreated on the mRNA and protein expression of ACE2(P=0.071 and P=0.188).High concentration of atorvastatin(10umol/L) had a more potent effects than the low concentration(1umol/L)(P＜0.001).ConclusionsThrough experiments of the above two parts,we can make the following conclusions:1.Oxidized LDL downregulates the mRNA and protein expression of ACE2 in both concentration and time dependent manner.2.Atorvastatin alone had no significant effects on gene and protein expression of ACE2 in HUVECs in both different concentration and different time.3.Atorvastatin,which blocks ox-LDL-mediated downregulation of ACE2 may exert beneficial effect in the prevention and treatment of atherogenesis.