The Regulation Of Macrophage Migration,Phagocytosis,Inflammation And Bone Marrow Mesenchymalstem Cell Proliferation,Migration By Wip1

With the improvement of the living standard,human health is threatened by a growing number of chronic diseases.Therefore,strategies regarding how to enhance self-repairing capability of the body become a hot research area.As one of the central topic in chronic diseases,atherosclerosis and its pathogenesis were extensively studied in a variety of animal models,but the underlying mechanisms of atherosclerosis remain largely undertemined.Procedures for therapy of atherosclerosis include removing intravascular lipids or restoring of vascular necrosis.In the human body,macrophages and bone marrow mesenchymal stem cells are the most important cells,involved in these two processes.These two cells to vascular disease from other body parts at play to their function involves multiple biological processes.These two cells migrate from other parts of the body to vascular disease and play at their functions involve multiple biological processes,including macrophages migrate in the vessel wall,lipid phagocytosis,trigger an inflammatory response and bone marrow mesenchymal stem cells(BMMSCs)proliferation and migration in blood vessels.Macrophages and bone marrow mesenchymal stem cells in these acts are cellular energy metabolism,including a variety of signaling protein phosphorylation and dephosphorylation process.Wip1,a newly discovered serine/threonine phosphatase,was reported to dephosphorylate multiple signaling transduction kinases.Previous studies found that the wipl-knockout can reduce the symptoms of atherosclerosis.It is great significance that made clear the molecular mechanism of this physiological process and research on new therapies targeted of atherosclerosis for future.This research selected wipl-WT and wip1-/-mice as experimental animals,and investigated the molecular mechanisms of Wip1 in regulating migration,phagocytosis,and inflammation of macrophages,as well as the contribution of Wip1 to cell proliferation and migration in bone marrow mesenchymal stem cells.The study focused on the following aspects:(1)Perform in vivo and vitro experiments to identify the signaling pathways required for the regulation of Wip1 on macrophages migration;(2)Detect the effects of Wip1 on pseudopod formation in macrophages;(3)Investigate the involvement of Wip1-related pathways in macrophage phagocytosis;(4)Measure the impact of Wip1 on inflammatory of macrophage;(5)Examine the effects of Wip1 on cell proliferation in cultured bone marrow mesenchymal stem cells;(6)Determine the influence of Wip1-related pathways on migration of cultured bone marrow mesenchymal stem cells.Based on the findings above,several conclusions could thereby be arrived as follows:(1)Wip1 knock out up-regulates Racl-GTPase activity and pAKT expression in macrophages,which in turn promotes macrophage migration in vivo and vitro.Cell polarization and filopodia formation are connected with Racl activity.The phosphorylation of AKT in PI3K/AKT pathway facilitates migration of macrophages.Wip1 knock out induces the activation of Racl-GTPase,which may also promote macrophages migration.(2)Negative feedback regulation of PI3K/AKT pathway by Wip1 in macrophages.PI3K/AKT antagonist down-regulates Wip1 expression,while activation of this pathway increase Wip1 level in macrophages.Compared with wild-type cells,the phosphorylation of AKT increases in Wip1 null macrophages.Therefore,Wip1 in macrophages may act on PI3K/AKT pathway in a negative feedback manner.However,whether Racl makes any difference in the above process needs to be further studied.(3)Wip1 mediates the expression of macrophage scavenger receptor(CD36)through AMPK pathway.When equal amount of wipl-/-and Wip1-WT macrophages is selected under flow cytometry,more wip1-/-can be screened,Flow cytometry detection of specific antigens on the surface of macrophage shows that CD36 expression in wip1-/-macrophages is higher than that in wipl-WT macrophages.Immunoblot assay also displays higher expression of pAMPK and CD36 in Wip-/-macrophages.The expression of CD36 protein is positively correlated with AMPK activity in macrophage.(4)Wip1 knock out triggers inflammation in macrophages.When inflammation occurs,macrophages release inflammatory factors.After a stimulation of inflammation with LPS for 1h,3h or 6h in macrophages,the levels of inflammatory cytokines(TNF-?,IL-6,IL-1?)in supernatant are higher in Wip1-/-macrophages.These data indicate that Wip1 knock out may promote inflammation in macrophages.(5)Wip1 knock out inhibited proliferation in cultured BMMSCs,leading to premature aging appearance.According to the records of BMMSCs passage time and cell proliferation,it is found that the growth of Wip1-/-BMMSCs slows down after the third generation.Cell cycle analysis shows G2 arrest in Wip1-/-BMMSCs.As a key regulator for the G2/M transition,Cyclin B1 initialize mitosis by forming a complex.Westernblot assay and immunofluorescence analysis show a very low expression of CyclinB1 in Wip1-/-BMMSCs.After 6 passages,most of the wipl-/-BMMSCs cease dividing,but display a growing cell volume and nuclei size.?-galactosidase staining indicates that the onset of premature aging after 5 passages will occur in most of the cells with 6 passages.(6)Wip1 knock out may induce migration of cultured BMMSCs through PI3K/AKT/Racl pathway.According to scratches and transwell assay,wip1-/-BMMSCs show a stronger migration ability than the wildtype ones.Wip-/-BMMSCs also display a higher pAKT level and Racl-GTPase activity.However,cell migration is terminated when blocking either PI3K/AKT pathway or Racl activity.Unlike macrophages,the inhibitor of PI3K/AKT reduces Racl activity in BMMSCs.These results suggest that Wip1 may regulate migration of BMMSCs via PI3K/AKT/Racl signaling.This research provides new evidence for Wip1's effects on migration,phagocytosis,or inflammation of macrophages,as well as its role in migration or proliferation of bone marrow mesenchymal stem cell.These findings enhanced the understanding about the pathogenesis of atherosclerosis,and raise a possible therapeutic regiment of atherosclerosis by targeting Wip1.