CXCL14 Functions As One Ring Of The Positive Feedback Loop In Atherogenesis

Background:Atherosclerosis is a vascular disease characterized by chronic inflammation of the blood vessel wall.The recruitment of monocytes into the subendothelial space of the arterial wall and the formation of foam cells are important conditions for the formation of early atherosclerotic plaques.In the CXC chemokine family,the relationship between the CXCL12/CXCR4 signal axis and the pathogenesis of atherosclerosis has been long-established,and this signal axis is thought to exacerbate various inflammatory diseases associated with immune cell recruitment.CXCL14/BRAK is a novel immune cell chemotactic agent of the CXC family with a structure similar to CXCL12,and it is not known whether it is related to the pathogenesis of atherosclerosis.Objective:The aim of this study was to investigate the expression and possible regulatory mechanisms of CXCL14/BRAK protein in atherosclerotic plaques,and to further investigate its role in the progression of atherosclerosis,providing a theoretical basis for finding potential therapeutic targets.Method:1.The expression of CXCL14 in atherosclerotic plaques of patients with atherosclerosis was tested by Immunohistochemistry and immunofluorescence.2.In vitro,the expression of CXCL14 in foam cells was detected by real-time PCR and Western blotting respectively in mouse primary macrophages and PMA(Phorbol12-myristate 13-acetate)-induced THP-1 cells.3.The activity of the AP-1 after stimulation with ox-LDL was detected in mouse primary macrophages by western blot and real-time PCR,and the direct interaction between AP-1 and CXCL14 promoter was demonstrated by luciferase reporter gene assay.4.Transwell and oil red O staining were used to detect the effect of recombinant CXCL14 on the migration and phagocytosis of THP-1 cells.5.The effects of recombinant CXCL14 on the migration and proliferation of smooth muscle cells were detected by scratch assay and CCK-8.6.After stimulating by recombinant CXCL14,western blot was used to detect changes of ERK phosphorylation in THP-1 cells and smooth muscle cells,and the effect of ERK phosphorylation on the above cell effects was also examined.7.In an animal model of atherosclerosis,anti-CXCL14-peptide was injected,and arterial blood vessel damage and plaque size of each group were examined to evaluate the lesion level.Result:Through the collection of clinical information and immunohistochemical staining of tissue samples,we found that CXCL14 was highly expressed in atherosclerotic plaques and is mainly expressed in macrophage-derived foam cells.The ox-LDL(oxidized low-density lipoprotein)stimulation can increase the expression of CXCL14 in macrophages in vitro.We studied the expression and activity of transcription factor AP-1stimulated by ox-LDL in mouse primary macrophages.The two subunits of AP-1(c-jun and c-fos)were increased in different degrees.The phosphorylation level of the c-Jun amino-terminal kinase(JNK)also increased,and when the activation of c-jun was blocked by SP600125(an inhibitor of the JNK pathway)the expression of CXCL14 was inhibited.Using luciferase reporter gene experiments,it was demonstrated that AP-1could directly bind to the promoter region of CXCL14.On the other hand,transwell experiments indicated that recombinant CXCL14 could promote the recruitment of THP-1 cells,while oil red O staining results indicate that CXCL14-treated macrophages(which are induced by PMA)are more capable of phagocytizing lipids.The scratch test was used to detect the effect of CXCL14 on the migration of smooth muscle cells.CXCL14 significantly promoted the migration of smooth muscle cells.At the same time,CXCL14 also slightly promoted smooth muscle cell proliferation,which also affected the formation of atherosclerotic plaques.THP-1 cells and smooth muscle cells were stimulated with CXCL14 at different times,and the phosphorylation level of ERK increased significantly,reaching a peak in about 10 minutes.Besides,when THP-1 cells and smooth muscle cells were pretreated with fr180204(ERK pathway inhibitor),the CXCL14 promotion of related cell functions was attenuated.In the animal model of atherosclerosis,anti-CXCL14-peptide was injected,area of the injured arterial blood vessel was significantly decreased compared with the control group.A full-thickness section of the aortic arch was taken and the plaque size was evaluated.The anti-CXCL14 group had smaller plaques than the control group.This also more intuitively reflects the important role of CXCL14 in atherosclerosis.Conclusion:Ox-LDL up-regulates the expression of CXCL14 in macrophages through AP-1,while the highly expressed CXCL14 in plaque further promotes the recruitment of monocytes and the phagocytosis of macrophages.It also promotes the migration and proliferation of smooth muscle cells.The positive feedback loop of CXCL14 promotes the formation of atheromatous plaques,and this providing a theoretical basis for CXCL14 to become a new target for the prevention and treatment of atherosclerosis.