The Role And Mechanism Of WKYMVm Inducing M2 Macrophages Polarization To Promote Angiogenesis In Bone Repair

Angiogenesis is essential for successful bone defect repair,while vascularization is a basic problem that needs to be solved urgently.In the early stage of bone healing,various repair cells are chemotactic and recruited,among which macrophages are the one of key regulators.With excellent plasticity,macrophages can effectively respond to environmental changes,switch phenotypes according to the surrounding signals and perform different functions.Generally,they can be divided into two main phenotypes according to the activation pathways: the classically activated M1 type and the alternately activated M2 type.M1 can produce a large number of pro-inflammatory cytokines to remove invasive pathogens and foreign bodies.M2 are mainly have anti-inflammatory effects and promote angiogenesis as well as tissue regeneration.Studies have shown that the expression of angiogenic growth factor and cytokines in M2 is higher than that in M1.Therefore,making full use of the plasticity of macrophages and making them polarize into specific subtypes in different environments has gradually attracted people's attention.Angiogenesis is primary need in bone repair,therefore,it is important to induce the M2 polarization to promote angiogenesis.WKYMVm(trp-lys-tyr-met-val-d-met-nh 2)is a hexapeptide identified by screening synthetic peptide libraries,which can specifically bind to formyl peptide receptor(FPR),which is mainly expressed in phagocytes.Studies have shown that WKYMVm exhibits immunoregulatory ability,especially immunosuppressive activity,in different physiological and pathological conditions by binding to FPR2.Although WKYMVm has been widely studied as an effective immunoregulatory peptide in the treatment of other diseases,whether WKYMVm can regulate macrophages to achieve its angiogenic function is still unknown,and the underlying regulatory mechanism is not clear.Activation of JAK1/STAT6 signaling pathway is a classic pathway to achieve M2 macrophage polarization.JAK is activated to further integrate STAT6 phosphoric acid into the nucleus,and the post-entry STAT6 binds to the nuclear receptor PPAR to mediate M2 polarization.It has been found that in human lung cancer cells calu-6,WKYMVm can trigger JAK1/STAT6 pathway by binding to FPR2.Similarly,studies in other cells have shown that FPR2 combined with agonists can activate downstream signaling molecules such as JAK,PI3 K,AKT,mitogen-activated protein kinases(P38,ERK,JNK),and finally promote cell migration,phagocytosis,and transcriptional expression of inflammatory cytokines.However,whether WKYMVm can regulate M2 through JAK1/STAT6 activation has not been clarified.Therefore,this study will explore the possible mechanism of WKYMVm inducing macrophage polarization.The purpose of this study was to clarify whether WKYMVm regulates M2 polarization to promote angiogenesis.And what is its potential mechanism of inducing macrophage polarization? In addition,in vivo,whether WKYMVm indirectly reshape and form new blood vessels by inducing M2 macrophage polarization,which is conducive to bone regeneration? Bases on the above hypothesis,we carried out the following work:1)Bone marrow macrophages(BMMs)were isolated as M ?.RAW264.7 cells and BMMs were treted with different concentrations of WKYMVm.After processing,flow cytometry and immunofluorescence were respectively performed to identify subtype of macrophages;real-time qPCR as well as Western blot were used for detection the expression of the relevant specific molecules of two subtypes of macrophage,so as to explore WKYMVm impact on macrophage polarization in vitro.2)The expression and secretion of PDGF-BB as well as VEGF in macrophage after WKYMVm induction were detected by real-time qPCR and ELASA.Then macrophage conditioned medium was colleted after WKYMVm pretreatment and used to incubate umbilical vein endothelial cells(HUVEC)for educating the scratches,lumen formation experiments.It is further verify the ability of WKYMVm-induced macrophage in promoting angiogenesis.3)The phosphorylation levels of JAK1 and STAT6 and the corresponding protein expressions in macrophage after WKYMVm stimulation were detected by real-time quantitative qPCR and western blot experiment.Then the possible molecular mechanism was speculated.In addition,in order to validate this pathway,Ruxolitinib a JAK1 phosphorylation inhibitor was added on the basis of wkymvm-induced incubation.Then followed by repeated real-time PCR and Western blot experiments to identify the macrophage subtypes,and repeated luminal formation experiments to detect the changes in the angiogenesis function of macrophages under the influence of inhibitor.4)In vivo,bone defect model in mice was established.The bone defects were treated with WKYMVm soaked gel sponge for interventional treatment.Two weeks later,the angiogenesis was observed by immunofluorescence staining.M? and M2 specific membrane proteins were double stained by immunofluorescence staining to observe the recruitment and polarization of macrophages;After 8 weeks,bone regeneration was observed by microscopic CT scan.At the same time,we used the chlorophosphate liposome targeting M? to clear macrophages and control a single variable as a control,excluding the direct effect of WKYMVm in vivo.Our results showed that: 1)When RAW264.7 cells or BMMs were treated with WKYMVm,they were induced to polarize to M2 instead of M1 macrophages.The M2 macrophage markers(CD206,arg-1,YM1,and IL-10)were up-regulated,while the M1 macrophage markers(CD86,iNOS,TNF-,and IL-1)were down-regulated.2)WKYMVm increased the expression and secretion of PDGF-BB in macrophage,and the corresponding WKYMVm-induced macrophages significantly enhanced the endothelial angiogenesis.3)WKYMVm activatied the phosphorylation of JAK1 and STAT6 and WKYMVm induces M2 macrophages polarization to promote angiogenesis through the JAK2/STAT6 pathway.Once the pathway was blocked,M2 will not be polarized and they will lose its angiogenetic function.4)In terms of bone defect repair in vivo,WKYMVm recruits macrophage,up-regulates the M2/M0 ratio at the defect site and induces the formation of new blood vessels to promote bone regeneration,but the chloropronate liposomes targeting depleted M? almost completely eliminate this process.In summary,our study showed that WKYMVm induces polarization of M2 macrophages in vivo and in vitro and in the end leading to angiogenesis.We further clarify that the molecular mechanism underlying these phenomena is based on the activation of the JAK1/STAT6 pathway mediated by WKYMVm.It suggests that WKYMVm may have an important regulatory effect on the macrophage phenotype,and these findings provide new ideas for vascularization in bone defects.