Essential Contribution Of Macrophage Tie2 Signaling In A Murine Model Of Laser-induced Choroidal Neovascularization

BackgroundAge-related macular degeneration(AMD)is the leading cause of irreversible visual impairment in the elder over 60 years old,and its incidence increases with age.Although,the exact etiology has not yet been clear,researchers believe that long-term light damage,genetic factors,metabolic factors were related with AMD.According to the clinical manifestations,it is generally divided into dry AMD(atrophic or non-neovascular AMD)and wet AMD,which was also known as exudative or neovascular AMD.Choroidal neovascularization(CNV)is a terminal symptom of wet AMD.Currently,the primary treatment for wet AMD uses VEGF.However,in addition to the economic burden of using the anti-vascular endothelial growth factor(VEGF)strategy,anti-VEGF therapy resistance represents a significant treatment challenge,prompting the need to identify a new,more efficient therapy.CNV which is directly related to aging and chronic stress diseases.Inflammation plays an important role in the neovascular stage of AMD(nvAMD).It was suggested that AMD is triggered by chronic low-grade,whole body and local inflammatory response.In this degenerative disease,deposits within the lesion contain many inflammatory mediators.Furthermore,AMD is associated with local inflammatory response as well as systemic activation of innate immunity.This inflammatory response includes activation of monocytes and descendant macrophages.Anti-inflammatory therapy is another potential option that can be used to treat AMD.These immunity activations have been found to manifest as activation of complement,mononuclear cell recruitment,and macrophage descendants.Inflammatory-related genes expressed in monocytes and peripheral blood mononuclear cells have been reported in nvAMD patients.A significant number of macrophages have been detected in AMD human eyes,and they modulated the formation of CNV in a laser-induced CNV(LCNV)murine model.Tyrosine protein kinase receptor 2(Tie2)is a receptor of Angiogenin located on cell surface,which regulates angiogenesis,endothelial cell survival,proliferation,migration,adhesion,diffusion,recombine actin cytoskeleton,and maintain vascular quiescence.Depending on different microenvironment,Tie2 plays the role of activating or inhibiting angiogenesis.It expressed not only on endothelial cells,but also on the surface of macrophages.Tie2-expressing on macrophages(TEMs)are a subpopulation of macrophages.The presence and phenotype of this subset of macrophages has been confirmed in human blood.TEMs have been found to promote angiogenesis in remodel tissues and tumours.Deletion of TEMs was reported to inhibit angiogenesis in limb ischemia,hepatocellular carcinoma,and tumor relapse.Traditionally,TEMs have been reported to accumulate in the tumor microenvironment,especially after chemotherapy or radiotherapy.TEMs have also been found to increase in limb ischemia after muscle injury.They play an important role in revascularization,which is associated with angiogenesis and vascular reconstruction.Furthermore,researchers have reported that,potentially,increased recruitment of TEMs play a role in enhanced neovascularization.However,the actual role of TEMs is still unclear in AMD.Therefore,the present study was designed to investigate whether the mechanism for TEMs contributes to LCNV as a model of AMD.Part Ⅰ Expression of TEMs and its effects on laser-induced choroid neovascularizationObjective:To explore choroid expression level of Tie2+macrophage and the role of TEM in laser-induced choroid neovascularization(LCNV).Methods:C57BL/6 mice were selected as the background animals,and the CNV murine model was constructed by laser injury.The dynamic change of Tie2+macrophages expression in choroid was observed by flow cytometry in Od,1d,3d,5d,7d and 14d after laser injury.Macrophage Tie2 signal was knocked out by Cre-loxP system.LyzCre+;Tie2flox/flox mice were defined as TEM-KO group,and,LyzCre+;Tie2flox/+ mice were defined as control,and Tie2 kinase inhibitor(TKI)intravitreal injection group was set as the positive control group.In fundus angiogram(FFA)and choroid flat mounts,CNV area was decreased in TEM-KO than control,with no significant difference compared with TKI group 7 days after laser injury.Retinal/choroid/sclera samples were collected on 0d,1d,3d,5d,7d and 14d after laser injury,and infiltration macrophages were detected via flow cytometry.VEGF-A,basic fibroblast growth factor(bFGF),interleukin-10(IL-10)and phosphorylation v-akt murine thymoma viral oncogene were detected by RT-PCR.The expression levels of p-AKT,phosphorylation extracellular signal-regulated kinase(p-ERK)and cleaved caspase3 were tested by Western Blot.Then,in 0d,5d,7d and 14d after laser injury,the apoptosis rate of CD31+vascular endothelial cells was measured by annexin V/PI double staining.Results:Laser injury was induced choroid plexus of mice.In fluorescent-activated cell sorting(FACS)analysis,TEMs infiltrated the choroid plexus within 1d after laser injury,then gradually increased within 3d to 5d,and peaked at 7d.But no significant difference of intra-choroidal F4/80+cells infiltration between TEMs-KO and control mice.The CNV area of the irrigated area was significantly less in the TEMs-KO group and TKI group than the control group.The peak mRNA expression levels of VEGF-A and bFGF were less augmented in the TEMs-KO group in comparison to the control group.Simultaneously,the peak value of IL-10 expressed higher and appeared earlier in the TEMs-KO group than the control.Western Blot analysis also demonstrated that the protein expression level of VEGF-A peaked at 3d.The maximum value of VEGF-A was lower in the TEMs-KO group than the control.In Western Blot,the present study confirmed that the peak expression level of p-ERK and p-AKT were both significantly lower in the TEMs-KO group than the control.Meanwhile,TEMs knockout is responsible for vascular endothelium cells apoptosis in LCNV.Conclusions:Tie2+macrophages exhibited enhanced laser induced CNV through enhanced choroid angiogenic factor expression and impaired apoptotic maker expression.Tie2 deletion on macrophages resulted in decreased LCNV.Part Ⅱ The effects of TEMs on mouse arterioles vessel endothelial cellsObjective:To address the effects and mechanism of Tie2 signaling on macrophage in capillary tube formation of mouse brain arterioles vessel endothelial cells(b.End3).Methods:TEM-KO mice were constructed with Cre-loxP system.Peritoneal macrophages from TEM-KO mice and control mice were stimulated and harvested.To observe whether the TEMs knockout affected macrophages polarization,the percentages of CD86+F4/80+cells and CD206+F4/80+cells were detected by flow cytometry.Peritoneal macrophages were cultured in vitro,then macrophage supernatants were collected.B.End3 was cultured with different macrophage supernatants.Under normal and hypoxia condition,mRNA expression level of VEGF-A,bFGF,IL-la and IL-10 were detected via RT-PCR.The effect of TEM in capillary tube formation,cell proliferation and migration of b.End3 was detected using three-dimensional matrigel assay,scratch assay and transwell assay respectively in vitro.The effect of TEMs in b.End3 expression of p-AKT,p-ERK and VEGF-A was examined with Western Blot.Cell apoptotic rate in b.End3 was examined using Annexin V/PI double-staining flow cytometry and caspase3 expression using Western Blot.Results:Macrophage Tie2 signaling had no effect on macrophage polarization.Hypoxia unregulated the expression of angiogenesis related factors,VEGF-A,bFGF,IL-1α,and IL-10.Tie2 deficiency did not reverse the mRNA expression trends in the MTie2+ and MTie2-group under the hypoxia condition.TEMs promoted the migration and proliferation ability of b.End3 cells in Scratch and Transwell assay.The number of tubes formed by b.End3 in MTie2-和 MTie2+ was remarkably more than that in DMEM group.Time-dependent kinetics of b.End3 apoptosis on annexin V/PI dual-staining demonstrated that exposure to the macrophage supernatant increased b.End3 apoptosis.Meanwhile,cleaved-caspase 3 protein level was significantly enhanced in the MTie2-group in comparison to the MTie2+ group after macrophage supernatants incubation for 12h and 24h.Conclusions:TEMs can increase b.End3 capillary tube formation through promoting cells migration,proliferation and expression of VEGF-A,p-AKT,and impairing of expression of cell apoptosis.