Chitinase-3-like-1 Promotes M2 Macrophage Differentiation And Induce Choroidal Neovascularization In Neovascular Age-related Macular Degeneration

Purpose.Strong choroidal neovascularization(CNV)is the characteristic of age-related macular degeneration(AMD)which leading to irreversible acute vision loss.Although anti-VEGF drugs as the principle treatment have alleviated the symptom,most patient resulted with resistence and recurrence.New target is in eager to be found.Immunity dysregulation with polarized macrophage M2 differentiation is associated with the progression of neovascular AMD(n AMD),but mechanism is still unclear.So,It is essential to uncover the mechanism of macrophage polarization-mediated CNV for improved treatment.Here,we are particularly interested in functional and mechanistic regulation of Chitinase-3-like 1(CHI3L1),a secreted glycoprotein capable of inducing tumor angiogenesis and macrophage differentiation,in macrophage M2 differentiation and subsequent induction of angiogenesis the laser-induced mouse model of CNV.Methods.(1)Patients with n AMD with or without anti-VEGFA treatment were selected to test serum levels of CHI3L1.(2)C57BL/6J mice were subjected to laser injury of retina to establish a choroidal neovascularization model,and choroidal-RPE was collected for western blots and real-time PCR and immunofluorescence staining to assess the expression of CHI3L1 and macrophage infiltration and M2 polarization.(3)RAW264.7 cell lines in vitro were cultured to test polarization and mechanism by CHI3L1 with western blotting and RT-PCR?si RNA knock down and COIP.(4)RF/6A cells were cultured to test angiogenesis mediated by CHI3L1 and VEGF-A and inhibition of their antibodies.(5)Anti-CHI3L1(m AY),anti-VEGF-A or combination(Ig G,PBS as control)was injected into the vitreous of mice model immediately after laser injury,FFA and immunofluorescence staining,choroidal flat-mount,SD-OCT were used to evaluate vascular development and M2 macrophage formation.Results.(1)Serum levels of CHI3L1 from patients with AMD either treated or untreated with anti-VEGF-A antibody were highly elevated compared with normal controls.(2)In a laser-induced murine CNV model,M2 markers(Arg-1 and Ym-1)were up-regulated with vascularization developed from day 1 to day 7 while M1 markers(TNF? and Ym-1)were elevated only in first day(P < 0.001).(3)Stimulation of RAW264.7 cells with recombinant CHI3L1 committed cell transition from M1 to M2 polarization and increased expression of M2 markers and VEGF-A(P < 0.001)while M1 markers without significant changes.Si RNA of IL13-R?2 can inhibited M2 polarization and activation of MAPK/ERK and AKT signaling.(4)recombinant CHI3L1 induced endothelial cell RF/6A-mediated tube vascularization.VEGF-A and CHI3L1 synergistically reduced tube formation to 30% while their combination promoted.(5)m AY can inhibited CNV and M2 polarization while combination of anti-VEGF-A resulted in synergistic effects in vivo.Conclusion.(1)CHI3L1 elevated in n AMD serum and without relevant with antiVEGFA treatment;(2)M2 polarized macrophages participated with development of CNV and CHI3L1 play critical role;(3)CHI3L1 can induce tube formation;(4)CHI3L1 activated MAPK/ERK and AKT signaling by interacted with IL13-R?2 and induced M2 polarization.;(5)m AY can inhibit CNV formation and anti-VEGFA can enhance its effect.