Effect And Mechanism Of PD-1 On Development Of Vulnerable Atherosclerotic Plaque

Background and ObjectivesThe pathogenesis of coronary heart disease(CAD) is atherosclerosis(AS).There are strong evidences that atherosclerosis is a chronic inflammation disease.The nature and feature of atherosclerosis are different markedly during its development and progression. Besides blood vessel narrowing,the composition and characteristic of plaque can exert great effects on clinical manifestation and prognosis of CAD patients.The vulnerable plaque, characterized by a big necrotic lipid nuclus,thin fibrous cap and abundant inflammatory cells infiltration,once rupture and thrombosis,it can further reduce blood flow or lead to coronary artery blockade.Vulerable plaque is a major factor contributing to acute coronary syndrome(ACS),reversal and stabilization of vulerble plaque is a important therapeutic target in ACS clinical strategies.Facilitating the study associated with starting and reinforcement of inflammation process in plaque has great economic and social profits. During the process of atherosclerosis development,immune system and immunocytes play key roles.As major risk factors,hyperlipemia and hyperglycemia can induce immune response in vessel wall.Some researches have provided evidences that oxidated low-density lipoprotein(ox-LDL),heat shock protein(Hsp) andβ2-glycoprotein I have been regarded as antigens related to atherosclerosis.During the development and progression of atherosclerosis,the effects of different subgroups of T lymphocyte are variant,CD4⁺ helper T cell has a dominant effect on development of atherosclerosis.The artery lesions of ACS are composed of rich vulnerable plaques.As Th1 over differentiation, imbalance of Th1 and Th2 in local lesions and circulation results in inflammation aggravation,lead to plaque destabilization.As a recent discovered negative costimulatory molecule,programmed cell death 1(PD-1) is a receptor expressed on activated T cell,B cell and myeloid cell.It belongs to CD28 family,its ligands(PD-Ls) such as PD-L1 and PD-L2 belong to B7 superfamily,expressed on macrophage and dendritic cell.In chronic viral infection PD-1 has been upregulated on T cell,but whether or not PD-1 expressed on CD4⁺ T cell in atherosclerosis has little been known.Our hypothesis is:If PD-1 expression on T cell in atherosclerosis be blocked,the T cell function and plaque vulnerability might alter. In this study the expression of PD-1 in atherosclerosis was immunohistochemically investigated,then CD4⁺ T cell function was mesured and plaque composition was tested using monoclonal antibodies against PD-1.MethodsThe first part:56 CAD patients were included in the study,ustable angina(UA) 30 and stable angina(SA) 26 cases.Serum ox-LDL level,cutured supernatant interferon-γ(IFN-γ) and interleukin-4(IL-4) of peripheral blood monouclear cell(PBMC) were detected by enzyme linked immunosorbent assay(ELISA).Flow cytometry was used to detect the expression of CD4⁺ and CD4⁺PD-1⁺ on PBMC in peripheral blood,and compared with control group,to investigate the relationship between the expression of PD-1 on CD4+ T cell and the inflammation degree in CAD patient.The second part:Twenty C57BL/6J ApoE gene knock out(ApoE^-/-) mice and 8 wild type C57BL/6J mice were fed with high fat diet.Twenty-one weeks later,all the mice were executed for the collection of aortas which were dissected from aortic root to abdominal aorta.Then the aortic sections were imbedded with paraffin for HE staining and PD-1 immunohistochemistry,the plaque area and expression of PD-1 were mesured.Interfered with Simvastatin,the variation of plaque area and expression of PD-1 were observed.The third part:The animal models of AS were established,and one of the AS groups was interfered with anti-PD-1 antibody,then the atherosclerotic morphology was studied, proliferation capability and excreted cytokine of CD4⁺ T cell were mesured.The vulnerability of atherosclerotic plaque and PD-1/PD-Ls signal pathway were investigated in order to reveal the latent mechanism.Results1.The PBMC cultured supernatant IFN-γand IL-4 levels in peripheral blood were increased obviously in CAD patients compared with control group(P＜0.05),IFN-γand IL-4 levels in UA group were further higher than those in SA group(P＜0.05),serum ox-LDL had a similar result.The levels of IL-4 had no significant difference in three groups.2.The percentage of CD4⁺ T cell of PBMC was higher in CAD patients than that in control cases(P＜0.05).The extents of CD4⁺PD-1⁺ double positive expression on PBMC were both higher in UA and SA groups than that in control group(P＜0.05),it increased markedly in UA(compared with SA P＜0.05).3.There were significant atherosclerotic plaques in AS model,plaque area of AS group was more remarkable than that of contol group.There was more expression of PD-1 on plaque in AS group than that in control group(P＜0.05),the plaque area and expression of PD-1 reduced in simvastatin group.4.The plaque area was not different significantly in anti-PD-1 intervention group and AS group,but the atherosclerotic plaque was characterized with more lipid,a great necrotic core,thin fibrous cap and fewer collagen.The average optical density(AOD) of collagen in anti-PD-1 intervention group was fewer compared with AS group(P＜0.05).5.There were more CD4⁺ T cells infiltrated in atherosclerotic plaque in anti-PD-1 intervention group when examined by immunofluorescence method.The AOD value of CD4⁺ T cell infiltration in anti-PD-1 intervention group was higher compared with AS group((P＜0.05).6.Cultured for 72 hours,CD4⁺ T cells separated from spleen had more ³H-TdR incorporation,higher IFN-γand IL-4 levels in anti-PD-1 antibody intervention group compared with AS group(P＜0.05,respectively).These results indicated the CD4⁺T cell had increased capability to proliferation and secreting cytokines.Conclusions1.In UA patients,as a antigen associated with atherosclerosis,ox-LDL may activate CD4+ T cells to secret more cytokines,lead to increased inflammation.2.The expression of PD-1 on CD4⁺ T cell increase when inflammatory response is enhanced,indicate the inhibitory effect of PD-1 on CD4⁺ T cell activation.3.The expression of PD-1 increase on atherosclerotic plaque during development of atheroma in ApoE-/- mice.Simvastatin can decrease plaque area and expression of PD-1 reduced,indicated the role of PD-1 may be limiting and downregulating inflammation. 4.Anti-PD-1 antibody intervention can increase CD4+ T cell infiltration and decrease collagen composition in plaque,lead to progression of vulnerable plaque.5.Anti-PD-1 antibody intervention can enhance the proliferation capacity and increase the secreted cytokines of CD4⁺ T cell,indicated the effect of PD-1/PD-Ls signal pathway on down-regulation inflammation.