The Role And Mechanism Of Different Macrophage Subtypes Regulating Bone Marrow-derived Cells In Retinal Neovascularization

Background:Retinopathy of prematurity（ROP）is defined as a multifactorial retinal vascular abnormal proliferative disease that has become the primary cause of blindness in children worldwide.The major pathological change of ROP is retinal neovascularization（RNV）.This pathological process can lead to vascular occlusion and retinal ischemia,further resulting in excessive vascular leakage and vascular proliferation,eventually causing tractional retinal detachment and blindness.Previous studies have shown that the development of ROP involves two distinct mechanisms:vasculogenesis and angiogenesis.Bone marrow-derived cells（BMCs）follow vasculogenesis,migrating from the bone marrow to the anoxic retina via stromal cell-derived factor-1（SDF-1）and vascular endothelial growth factor（VEGF）.After activation in the retina,BMCs mainly differentiate into endothelial cells（ECs）and smooth muscle cells（SMCs）to participate in the pathology of RNV.Other studies have confirmed that macrophages can increase the chemotaxis of BMCs in the retina and promote the severity of RNV by up-regulating SDF-1 and VEGF.Macrophages are usually activated into two categories.The first is the classically activated and pro-inflammatory state,named M1macrophages.The second category,so-called‘alternatively activated’macrophages,are known as M2 macrophages.Both categories play an important role in the development of RNV.However,the role and mechanism underlying the regulation of BMCs by different macrophage subtypes during RNV remains unclear.It is,therefore,necessary to further study.ObjectiveTo investigate the role and mechanism of different macrophage subtypes that regulate BMCs during the development of RNV.Methods1.The role of different macrophage subtypes in RNVThe bone marrow-derived macrophages（M0）were cultured in vitro and induced to M1and M2 subtypes.RT-q PCR and Western Blot were used to identify whether the polarization was successful.C57BL/6J pups and their mothers were exposed to a high-oxygen chamber of（75±2）%from post-natal day 7（P7）to P12 to establish the oxygen-induced retinopathy（OIR）model.The M0,M1,M2 macrophages,or phosphate buffer saline（PBS）were injected into the vitreous at P12.The retinas of the mice at P17 in each group were performed with retinal flatmounts and lectin staining to analyze the severity of RNV.2.The regulation of different macrophage subtypes on BMCs in RNV（1）In vivo experimentsNeonatal C57BL/6J mice were irradiated with 2.0 Gry ⁶⁰Co at P1.Green fluorescent protein（GFP）-positive BMCs,derived from GFP transgenic mice,were transplanted into irradiated C57BL/6J mice at P1 to establish C57BL/6J-GFP chimeric OIR mice.Pups were killed at OIR-P12 or P17,and the retinas were performed with retinal flatmounts and lectin staining to investigate the participation of BMCs in RNV.The PBS,M0,M1,or M2macrophages were injected into the vitreous of chimeric mice at OIR-P12.Retinal flatmounts and lectin staining were used to analyze the recruitment of BMCs around the retinal neovascularization（NV）tufts at P17.Immunofluorescent staining of frozen retinal sections was used to analyze the differentiation（CD31-positive ECs andα-SMA-positive SMCs）of recruited BMCs around the retinal NV tufts at P17.（2）In vitro experimentsTo collect conditional medium（CM）from different subtypes of macrophages,the supernatant was removed after macrophages were polarized,and different macrophage subtypes were continued to culture with the medium of stem cells for 24 h.RT-q PCR and Western Blot were performed to identify the polarization of different macrophage subtypes after de-stimulation,and the conditional medium was then collected.They are divided into four groups:control group（standard medium of stem cells as control）,M0-CM group,M1-CM group,and M2-CM group.Bone marrow mesenchymal stem cells（BMSCs,an important subset of BMCs）were treated with the above four conditioned media,respectively.Transwell,RT-q PCR,and Western Blot were used to investigate the migration and activation（including the differentiation of BMSCs and the impact of degradation of the extracellular matrix）ability of BMSCs following stimulation with conditional medium from different subtypes of macrophages.3.The mechanism of the regulation of BMCs by the different macrophage subtypesRT-q PCR,Western Blot,and immunofluorescence were used to investigate the expression of SDF-1 and its receptor chemotaxis cytokine receptor-4（CXCR4）,as well as VEGF and matrix metalloprotein-9（MMP-9）in different macrophage subtypes and BMSCs cultured with the conditional medium of each subtype.Receptor antagonists AMD3100 or si CXCR4 was used to block the SDF-1/CXCR4 signaling pathway to observe the effect of polarized macrophages on the migration of BMSCs by Transwell assay.Results1.The role of different macrophage subtypes in RNVThe results of RT-q PCR and Western Blot showed that the expression of M1 and M2macrophage-specific markers was significantly increased（P<0.01）.M1 macrophage-specific markers,including inducible nitric oxide synthase（i NOS）and interleukin-1β（IL-1β）,and of M2 markers,including arginase-1（Arg-1）,Ym1,and mannose receptor（MR）.The OIR model was established and the PBS,M0,M1,or M2 macrophages were injected intravitreally at OIR-P12 mice.OIR-P17 retinal flatmounts and lectin staining revealed that the retinal avascular area and NV tuft area in the M2 macrophage group were more extensive than those in the PBS,M0,and M1 macrophage groups（P<0.01）.2.The regulation of different macrophage subtypes on BMCs in RNV（1）In vivo experimentsThe C57BL/6J-GFP chimeric OIR model was established,and retinal flatmounts and lectin staining revealed that BMCs could be recruited around the retinal avascular area at P12 and in the NV tufts at P17.Then PBS,M0,M1,or M2 macrophages were injected intravitreally at chimeric OIR-P12 mice,and P17 retinal flatmounts and lectin staining revealed that the number of GFP-positive BMCs in the M2 macrophage group was higher than that in the PBS,M0,and M1 macrophage groups（P<0.001）.Immunofluorescent staining of frozen retinal sections showed that the degree of co-expression of GFP-positive BMCs and CD31-positive ECs orα-SMA-positive SMCs in the M2 macrophage group was higher than that in the PBS,M0,and M1 macrophage groups.（2）In vitro experimentsRT-q PCR and Western Blot showed that M1 and M2 macrophages still expressed their respective specific markers after the removal of polarizing factors for 24h.The specific markers of M1 macrophages include i NOS and IL-1β,and the specific markers of M2include Arg-1,Ym1,and MR（P<0.05）.Then the BMSCs were treated with conditional medium from different subtypes of macrophages,which were divided into four groups,including the control,M0-CM,M1-CM,and M2-CM groups.A Transwell assay showed that the number of migrating BMSCs in the M0-CM and M2-CM groups was higher than that in the control and M1-CM groups（P<0.01）.RT-q PCR showed that the expression of CD31,vascular endothelial-cadherin（VE-cadherin）,von Willebrand factor（Vwf）,α-SMA,SM22,and cathepsin L m RNA in BMSCs cultured with M2-CM was higher than that in the M1-CM group（P<0.01）.CD31,VE-cadherin,and Vwf are markers of ECs,whileα-SMA and SM22 are markers of SMCs,and the degradation of the extracellular matrix is regulated by cathepsin L.Western Blot revealed that M2-CM also increased the protein expression of CD31,VE-cadherin,α-SMA,and cathepsin L compared to that of M1-CM（P<0.05）.3.The mechanism of the regulation of BMCs by the different macrophage subtypesThe RT-q PCR analysis showed that the relative expression of SDF-1 and its receptor CXCR4,as well as VEGF and MMP-9 m RNA,was higher in M2 macrophages than in M1macrophages（P<0.001）.The protein expression of these factors in M2 macrophages was also higher than that in M1 macrophages by Western Blot and immunofluorescence（P<0.05）.Similarly,RT-q PCR,immunofluorescence,and Western Blot also confirmed that BMSCs cultured with M2-CM expressed a higher level of SDF-1,CXCR4,VEGF,and MMP-9 than those cultured with M1-CM（P<0.05）.Finally,a Transwell assay showed that the migration of BMSCs was reduced by adding AMD3100 or si CXCR4 to block the SDF-1/CXCR4 pathway（P<0.05）.ConclusionsThese data demonstrate that M2 macrophages play a more important role than M1macrophages in promoting the development of RNV.Our results also indicate that M2macrophages may express significantly higher levels of SDF-1,CXCR4,VEGF,and MMP-9 than M1 macrophages,thus regulating the recruitment and differentiation of BMCs and further aggravating vasculogenesis during RNV.We propose that a comprehensive understanding of the role of M1 and M2 macrophages during pathological RNV,especially the role and mechanism underlying the regulation of BMCs by different macrophage subtypes,may yield potential therapeutic targets for the treatment of RNV.