The Role And Mechanism Of TLR4 Signaling Pathway In Arterial Neointimal Formation

Background: Arterial neointimal formation,that causes arterial stenosis and eventually leads to tissue ischemia and hypoxia,is the main cause of heart failure and graft failure.The underlying mechanism of arterial neointimal formation is not well known.Toll-like receptor 4(TLR4)is an important component of innate immunity.Though its role and disease in infectious diseases have been well clarified,the role and potential mechanism of arterial neointimal formation have not yet been elucidated.Objective: To explore the role of TLR4 signaling pathway in the formation of neointima of mouse ateries as well as its molecular mechanisms,and to find possible therapeutic targets.Methods: First part 1.A mouse aortic allograft model was established.BALB/c(H-2d)donor aortas weretransplanted into C57BL/6(H-2b)recipients to detect graft-associated pathological changes and the expression of TLR4.2.TLR4 inhibitor intervention: TAK-242,a TLR4-specific inhibitor,was administered to the mouse vascular graft model,1 day prior to surgery,at the day of surgery then every other day for 8-12 weeks.3.Detection methods and indices:(1)Immunohistochemical(IHC)and immunofluorescence(IF)staining were used to detect the infiltrating inflammatory cells and TLR4 expression in the grafts;(2)The expression of TLR4 pathway was detected by RT-q PCR and Western blotting;(3)ELISA was used to detect the expression of inflammatory factors and chemokines in the supernatant of cell culture medium;(4)The proportion of macrophages was detected by flow cytometry;(5)Establish a co-culture system and cell chemotaxis assay to confirm the target.Second part 1.A mouse model of intimal hyperplasia/arterial remodeling was established.The proximal common carotid artery and the bifurcation of the carotid artery were sutured with silk thread,and the pathological changes of the injured vessel were detected.2.CD40 antibody and CD40/TRAF inhibitor intervention: The mouse intimal hyperplasia/artery remodeling model was given CD40 antibody or CD40/TRAF inhibitor 6877002 for 4 weeks.3.Detection methods and indices:(1)Immunohistochemical(IHC)and immunofluorescence(IF)staining were used to detect the infiltrating inflammatory cells in the injured vessels;(2)The expression of MMPs was detected by RT-q PCR and collagenase activity assay;(4)The proportion of T cells,B cells and macrophages was detected by flow cytometry;(5)Effectiveness of the drug was detected in cultured bone marrow-derived macrophages.Result: First part 1.This study successfully established a mouse model of aortic allograft transplantation.Vascular intimal hyperplasia of allografts was significantly greater than that of isografts.Infiltration of inflammatory cells was also significantly more than isografts.TLR4 is involved in the intimal hyperplasia of a mouse model of aortic transplantation and primarily localized to macrophages.2.The TLR4 inhibitor TAK-242 reduced intimal hyperplasia in allograft and delayed the formation of neointima;TAK-242 reduced the infiltration of CD68+ macrophage in grafts by downregulating Ly6 Chi macrophage in peripheral blood,bone marrow and spleen after transplantation.3.The TLR4 inhibitor TAK-242 impaired the IL12p70/IFNγ axis in a mouse vascular graft model by inhibiting the NF-κB signaling pathway,thereby reducing the expression of pro-inflammatory cytokines and chemokines in the neointimal of the graft and decreasing the CCL2-mediated vascular smooth muscle migration.Second part 1.This study successfully established a model of mouse intimal hyperplasia/arterial remodeling.Blocking CD40,neointimal formation were attenuated,and activities of gelatinase and collagenase in neointimal formation and arterial remodeling are reduced.2.CD40/TRAF6 inhibitor 6877002 significantly attenuated neointimal formation,reduced inflammatory infiltration and collagen conversion,significantly inhibited M1 polarization,and induced generation of IL-10-producing regulatory macrophages.Conclusion: 1.TLR4 is involved in neointima formation in mouse aortic transplatation model andmouse intimal hyperplasia/arterial remodeling model.The main mechanism may be:(1)participate in the polarization of macrophages and promote the secretion of inflammatory factors;(2)promote the self-circulation of IL12p70/IFNγ axis,and further enhance the inflammatory response and tissue repairing.2.TLR4 inhibitors and CD40/TRAF6 inhibitors can significantly reduce the vascular lesions in the two neointimal formation models,respectively.