Effects Of Oxidized Low Density Lipoprotein And Simvastatin On The Expression Of PD-L1 In Human Vascular Endothelial Cells

BackgroundCardiovascular disease caused by atherosclerosis is a common disease damaging people's health. Ross thought atherosclerosis is a chronic inflammatory caused by injury factors including oxidative low density lipoprotein (ox-LDL). 1t is quite complex for AS which formed by the cooperated effects of infection, inflammation, and immunity.The activation of T cells is most important for AS. T cells require two signals:the identify signal and the co-stimulating signal to proliferate and differentiate into effector cells. Antigen-specific activation and proliferation of T lymphocytes are regulated by both positive and negative signals from the B7 family of costimulatory molecules.PD-L1 have been identified in the past years,which play important role in regulating T cell responses.Statins are potent inhibitors of 3-hydroxy-3-methylgl-utaryl coenzyme A reductase. These drugs are capable of lowering the serum levels of cholesterol and are successfully used to treat hypercholesterolemia and atherosclerosis.Moreover, the ability of statins to reduce the mortality and morbidity of cardiovascular diseases has been descri bed not only to their cholesterol lowering activities, but also to a number of additional effects, including improved endothelial cell function, enhanced fibrinolysis, and antithrombotic activity. In addition, a number of important anti-inflammatory effects of statins have been reported.The anti-inflammatory effects of statins implicated more clinical benefit that can be obtained in the treatmen of atherosclerosis. Little is known about the mechanisms by which statins counteract inflammation.Whether statins can regulate PD-L1 costimulatory signal in delaying the progress of AS, it is worth further study and exploration.Objective1,To determine the effects of ox-LDL on gene and protein expression of PD-L1 in HUVECs.2,To determine the effect of simvastatin on the mRNA and Protein expression of PD-L1 in HUVECs activated by ox-LDL.3,To discuss the relationship of ox-LDL,simvastatin,and PD-L1 in the mechanism of atherosclerosis.Subjects and Method1,Subjects:The 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 withlOmmol/mL CuSO4. The oxidation was stopped with 100μmol/L EDTA.Polyacrylamide gel electrophoresis identify ox-LDL.2.2 Preparation of simvastatinCrude drug of simvastatin(41.86mg) was dissolved in 2ml pure alcohol,0.1 mol/L NaOH of 1.8 ml was added.Then the mixture was heated at 80℃for 4 hours,and PH value was regulated with 0.1 mol/L HC1 to be 7.2,then PBS was added to 10 ml, and 10mmol/L reserved simvastatin solution was prepared and stored at 4℃.2.3 Culture and pasage of HUVECsThe freezing HUVECs lines were inoculated in M199 medium supplemented with 50 ng/L ECGS,5 kU/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 2-3 days with "cobblestone appearance" in optical microscopy, then 0.25% trypase were used in passage of HUVECs at recultured to subconfluence.Cells of 3-6th passage were initially trypsinized,transferred to 10cm2 culture dishes with the concentration of 2.0 X 106 cells/well and recultured 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 PD-L1(1) control group:HUVECs cultured with equal M199 medium as control;(2-4) Different concentration groups of ox-LDL:HUVECs were treated with different concentration ox-LDL(10mg/L,50mg/L and 100mg/L) for 24h,respectively;2.4.2 Effect of simvastatin on gene and protein expression of PD-L1 in HUVECs activated by ox-LDL.(1) control group:HUVECs cultured with equal M199 medium as control;(2) simvastatin group:HUVECs were incubated with 5μmol/L simvastatin for 24h.(3)simvastatin+ox-LDL group:HUVECs were preincubated with 5μmol/L simvastatin for 1h and then cocultured with 50mg/L ox-LDL for 24 h.(4) ox-LDL groups:HUVECs were cultured with 50mg/Lox-LDL for 24h as control. (5) IFN-γgroup:HUVECs were incubated with 100U/ml IFN-γfor 24h.2.5 Semiquantitative RT-PCR for PD-L1:PD-L1 mRNA was examined by RT-PCR.Total RNA extracted from cultured HUVECs was reverse-transcripted using specific primers for human PD-L1.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 FACS for PD-L1:HUVECs were collected,and analysed the expression of PD-L1 by FACS.2.7 Data analysis:SPSS 13.0 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 factorⅧrelated 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 the expression of PD-L1 on HUVECs.Incubation of HUVECs with ox-LDL(10mg/L,50mg/L and 100mg/L)for 24h significantly increased the expression of PD-L1(mRNA and protein)in a concentration-dependent manner(P<0.001).When compared with control group,PD-Ll mRNA expression increased to 148%,270%,393%(P<0.001),and protein expression increased to 120%,154% and 193%(P< 0.001),after treated with different concentrations of 10mg/L,50mg/L and 100mg/L ox-LDL.3,simvastatin affect the expression of PD-L1 on HUVECs excited by ox-LDL.Compared with the blank control group,50mg/L ox-LDL and 100U/ml IFN-γincrease PD-L1 expression on HUVECs significantly (P< 0.001). Preincubation of HUVECs with simvastatin significantly inhibited PD-L1 mRNA and protein induced by ox-LDL compared to 50 mg/L ox-LDL group(P< 0.001). Group which coculture with 10umol/L simvastatin is no significantly to control group on the mRNA and protein expression of PD-L1(P=0.838, P=0.3638, respectively)。Conclusions:1,Oxidized LDL up-regulates the mRNA and protein expression of PD-L1 in concentration dependent manner.2,Simvastatin alone had no significant effects on gene and protein expression of PD-L1 in HUVECs in 5μmol/L concentration.3,simvastatin, which blocks ox-LDL-mediated upregulation of PD-L1 may exert beneficial effect in the prevention and treatment of AS.