The Role And Mechanism Of Dickkopf-3 Ablation In The Development Of Atherosclerosis

Background and Objective:Dickkopf-3(DKK3)is a negative regulator of the Wnt/β-catenin signaling pathway,which is involved in inflammation.However,little is known about the relationship between DKK3 expression and the progression of atherosclerosis.To define the role of DKK3 and its potential mechanism in the development of atherosclerosis by using an ApoE-deficient mouse model and DKK3－deficient mouse model.Methods:(1)Construction of atherosclerosis in mice model:Mouse was divided into four groups,namely ApoE-/-NC,DKK3-/-ApoE-/-HFD,DKK3-/-ApoE-/-NC.Eight-week-old mice of 2 groups were treated with a high-fat diet(HFD;15.8%fat and 1.25%cholesterol)or normal chow(NC)for up to 28 weeks.Serum was collected for the measurement of lipids and the levels of inflammatory cytokines.Paraffin-embedded slice was used for Hematoxylin-eosin(HE)staining to quantify the size of atherosclerotic lesions and for immunofluorescence to detect Related protein expression levels the inflammation,while the molecular samples was prepared for western blot to detect investigate the change level of protein and signal pathway.(2)Bone Marrow Transplantation Study:Male recipient mice at 8 weeks of age were lethally irradiated,lethally irradiated ApoE-/-recipient mice were transplanted with bone marrow from DKK3-/-ApoE-/-or ApoE-/-donors and had atherosclerosis.Four weeks after bone marrow transplantation,peripheral blood was collected for polymerase chain reaction(PCR)analysis of bone marrow reconstitution.Then,mice were fed a HFD for an additional 16 weeks to analyze atherosclerotic lesion development.(3)In vitro experiment:in bone marrow-derived macrophages exposed to oxidized-LDL stimulation.obtainning peritoneal macrophages in mice after bone marrow transplantation,then exposed to oxidized-LDL stimulation for 24h,Extracted protein was used for the molecular samples.Results:(1)The expression:The expression of DKK3 in the right coronary artery of patients with coronary heart disease was higher compared with normal arteries group,immunofluorescence staining of human plaque also revealed significantly increased DKK3 expression in the macrophages of atherosclerotic arteries compared with normal arteries.Western blot revealed a strong increase of DKK3 expression in aortas from ApoEdeficient mice fed with HFD for 28 weeks compared with NC treatment.Immunofluorescence staining of the aortic root revealed a stronger immunoreactivity in the atherosclerotic lesion of HFD-induced ApoE-/-mice than the NC group.Additionally,the upregulated expression of DKK3 was found to be primarily localized in the lesioninfiltrated macrophages.(2)The area of atheromatous plaques strongly reduced and the plaque stability score increased significantly in DKK3-/-ApoE-/-mice compared with ApoE-/-mice.(3)Transplantation of bone marrow from DKK3-/-ApoE-/-mice into lethally irradiated ApoE-/-recipients resulted in a reduction of atherosclerotic lesions,compared with the lesions in recipients transplanted with ApoE-/-donor cells.(4)Inflammation:The mRNA levels of ICAM-1 and VCAM-1,2 endothelial adhesion molecules and proinflammatory markers such as IL-6,IL-lb,TNF-a,MCP-1 were strongly reduced in DKK3-/-ApoE-/-mice compared with ApoE-/-mice.Immunofluorescence staining showed decreased expression of ICAM-1 and IL-6 and reduced phosphorylation of the NF-κBsubunit p65 in the lesion area of DKK3-/-ApoE-/-mice.Western blotting showed that the expression levels of phosphorylated of IKKβ,IκBα,and p65 were decreased in DKK3-/-ApoE-/-mice compared with ApoE-/-mice.(5)Possible molecular mechanism:Western blotting analysis revealed a significant decrease of phosphorylated b-catenin but increased dephosphorylatedβ-catenin in the cytoplasm of DKK3-/-ApoE-/-mice compared with ApoE-/-ittermates.Meanwhile,dephosphorylated β-catenin in the nuclear fraction was strongly increased in DKK3-deficient mice compared with the control group.Immunofluorescence staining indicated the translocation ofβ-catenin in the nuclei of macrophages in DKK3-/-ApoE-/-mice,The similar results in accordance with experiment in vivo were also obtained in peritoneal macrophages isolated from ApoE-/-and DKK3-/-ApoE-/-mice treated with oxidized LDL.Conclusions——DKK3 expression in macrophages is involved in the pathogenesis of atherosclerosis through modulation of inflammation and inactivation of the Wnt/β-catenin pathway.