Involvement Of Basigin-2 In Microglia-mediated Regulation Of Oxygen-induced Retinal Angiogenesis

BackgroundRetinopathy of prematurity(ROP),especially,occurring in preterm and low birth weight infants,is the principal cause for blindness in children in the world.Supplemental oxygen therapy required for preterm infant survival,perturbs the normal development of immature retinal blood vessels and thus leads to abnormal vessel growth in vaso-obliteration areas in the immature retina and ultimately formation of hemorrhages and retinal detachment.Relative hypoxia induced angiogenesis is one of the crucial pathogenesis in ROP.Angiogenesis is the adaptive process organisms to hypoxic circumstances,including multiple steps involving the interactions between endothelial cells(EC)and multiple perivascular cells within microenvironment.Its mechanism is complicated and remains yet to be fully understood.Microglia cells,the resident macrophages cells of central nervous system(CNS),play pivotal roles in the immune response,development,tissue homeostasis and wound healing.Recently,a large number of studies have used the development of mouse retina as a model of angiogenesis.The results show that retinal microglia is the early driving force of angiogenesis.The close spatial localization of retinal microglia and blood vessels suggest an emerging role of microglia in vascular angiogenesis and vasculopathy.However,the molecular mechanism underpinning this process has not been fully elucidated.Moreover,it is worth noting that microglia,with a highly increased motility,can sense physiological and pathological cues,survey and communicate with the surrounding microenvironment.Microglia can respond swiftly to local chemotactic cues and affect surrounding neuronal and vascular components by secreting various cytokines and neurotrophic factors to accomplish intra-cellular communication.Thus,in ROP,microglia can bridge local hypoxia to an innate adaptive response in neovascular retinal disease.Basigin,an extracellular matrix metalloproteinase inducer(EMMPRIN,also called CD147),is a pleiotropic highly glycosylated transmembrane cell surface glycoprotein.While predominantly recognized for its role in multiple biological processes,such as immune response,tumor progression,matrix proteolysis and cell migration,it has also been shown to be involved especially in neovasculogenesis under hypoxia during tumor invasion and metastasis.Especially,it has been reported that basigin may promote angiogenic process not only through its protease-inducing function by up-regulating matrix metalloproteinase(MMPs)secretion,but also directly by its ability to increase soluble forms of VEGF and VEGFR-2 in tumor cells and EC.Studies have provided evidence that basigin can be released from ovarian cancer patients in microvesicles or exosomes,which can interact with surrounding microenvironment promoting tumor angiogenesis.Besides,recent studies have reported that expression of basigin was elevated in monocytes/macrophages and played a role on regulating the inflammatory activities of these cells.Basigin is widely involved in the formation of new blood vessels under hypoxic conditions.However,the role that basigin palys during the interaction between microglia and vascular EC in the ROP,in which hypoxia functions as one of instrumental pathological mechanisms,remains under study.ObjectiveIn this study,the role and mechanism of microglia in the regulation of hypoxic retinal neovascularization through basigin were discussed.The aim of this study was to provide experimental reference for the treatment of hypoxic ischemic retinal neovascularization.Methods1: Involvement of basigin in microglia-mediated promotion of oxygen-induced retinopathy related angiogenesis(1)Animal model: OIR mouse model was set up.(2)Immunohistochemistry methods: Retinas were harvested and sectioned or flat-mounted.Immunohistochemistry analysis and immunofluorescence double staining using basigin and IBA-1 were carried out to assess the location of microglia and the expression pattern of basigin in retina.Their co-localization was especially evaluated.(3)Western blot analysis: The variations of hypoxia inducible factor-α(HIF-1α)and basigin expression in the neural retina were analyzed at P12 and P17 of OIR model.2: Effects of basigin expressed by microglia on the angiogenesis capcity of chorioid-retinal microvascular EC(1)Cell model: The chemical hypoxia model was established in BV2 cells,a microglia cell line.(2)Co-culture systems: Co-culture systems of BV2 and chorioid-retinal microvessel EC(RF/6A)were set up respectively at direct contact or by conditional medium from BV2.(3)Western blot analysis:Cellular basigin,HIF-1α,IGF-1,AKT,P-AKT,ERK and P-ERK expression in BV2 under different treatments were quantified.Moreover,the expression patterns of VEGF and VEGFR-2 in RF/6A were also assessed.(4)q RT-PCR: The expression patterns of IGF-1,basigin-1 and basigin-2 isoforms in BV2 and the level of VEGF and VEGFR-2 from RF/6A were examined.(5)Endothelial cell migration assays: The migration capacity of cultured RF/6A was assessed by cell migration under Transwell chamber.(6)Endothelial cell tube formation assay: The angiogenic capacity of cultured RF/6A was evaluated by tube formation assay using Matrigel.The length of the tubes was measured using Image Pro Plus software.(7)Small interfering RNAs transfection: Basigin-2 in BV2 was knocked down by liposome encapsulated si RNA.(8)Cytokine quantification: Paired cytokine antibody arrays were used to examine the expression profiles of several releasable angiogenic factors up-regulated under hypoxia and down-regulated after basigin being knocked down in supernatant from BV2.(9)Inhibitor intervene: Related pathway antagonists including AKT inhibitor LY294002 or MEK inhibitor PD98549 were independently used to elucidate the possible signaling pathway involved in the enhancement of angiogenesis by basigin-2.Exogenous IGF-1 protein or IGF-1 antibody and IGF-1 receptor inhibitor were used to block or activate IGF-1.Then,the effect of RF/6A on tube formation was observed.(10)Statistical analysis: All statistical analyses were performed using SPSS 17.0.Multiple comparisons referring to more than two groups,a one-way ANOVA was performed,in conjunction with Student-Newman-Keuls(SNK)test.A value of P < 0.05 was considered statistically significant.Results1.In vivo,basigin was overexpressed in retinal microglia near angiogenic sprouts in OIR mouse model.In contrast with control normal mouse,data from sectioned or flat-mounted retina using immunofluorescence staining both revealed significantly higher numbers of IBA-1+ microglia localized in close proximity to endothelial sprouting pathological tufts during neovascularization.Moreover,the expression of microglia marker IBA-1 and basigin were significantly co-localized.The variation of basigin expression in neural retina was assessed by western blot.The mean value of OIR in group P17 was higher than that in normal control group(P=0.0012).2.In vitro,effects of microglia expressed basigin-2 on the angiogenesis capcity of retinal vascular endothelial cells and the underlined mechanism were investigated.1)Up-regulation of basigin-2 and HIF-1α expression in BV2 cells under hypoxic condition.The expression of basigin and HIF-1α increased in a time dependent manner after hypoxia treatment as shown by western blot.The expression of basigin in hypoxia 12 h group(P=0.0097)and 24 h group(P=0.0003)were both higher than control group.Results from q RT-PCR revealed that basigin-2 was the predominant basigin isoform expressed by hypoxic microglia,and increased with the extension of hypoxia time.2).Microglia under hypoxia treatment facilitated tube formation of microvascular EC.Tube formations of RF/6A were enhanced compared to controls either during co-cluture with microglial cells or under conditioned medium from microglial cells after hypoxia treatment.At 6h after inoculation,the length of tube formation in the co-culture group was 60.80% higher than that of the control group(P=0.0001).The mean of the conditioned medium group increased by 52.37%(P=0.0003).However,there was no significant difference between the co-culture group and the conditioned medium group (P=0.4008).Thus,the role of microglia played in facilitating tube formation is not necessarily dependend on direct cell-to-cell contact.3).Liposome encapsulated si RNA significantly silenced basigin-2.Quantitative RT-PCR and Western blot were used to validate the effect of liposome coated si RNA knocking down on basigin-2.Both si RNA 317 and 458 sequences could significantly down-regulate the expression of basigin protein and nucleic acid expression.However,the inhibitory effect was better when two si RNA sequences combined to interfere with basigin-2.4).Silencing basigin-2 expression in microglia inhibits the angiogenic capacity of retinal EC.The angiogenic capacity of retinal EC was inhibited when treated with conditioned medium from hypoxic BV2 after silencing basigin expression.After basigin-2 being knocked down in BV2,the number of migrating cells decreased by 65.93%(P=0.0098);and the length of tube formation reduced by 68.27%(P=0.0001).5).Silencing microglial basigin-2 expression inhibited production of proangiogenic factors.Cytokine array analyses evaluated the change of proangiogenic factors after silencing microglial basigin expression.Four factors including IGFBP2,IGF-1,Pro-MMP9,and VEGFR-1 were up-regulated in hypoxia after hypoxia,and decreased after basigin-2 being knocked down.The change of IGF-1 expression was modulated maximally,which was increased 3.11 times after hypoxia,and then decreased 1.33 times.After basigin-2 being knocked down,the level of IGF-1 m RNA decreased(P=0.0001);and the protein expression also down-regulated(P=0.0082).6).IGF-1 enhanced tube formation in EC.IGF-1 neutralizing antibody or exogenous IGF-1 was applicated in conditioned medium independently,combined with microglial basigin-2 silence by si RNA,to evaluate the effect of IGF-1 on tube formation.Six h after inoculation,in mock-knocked down together with IGF-1 antibody group,tube formation decreased by 28.83%(P=0.0098);while in basigin-2 knocked down by si RNA together with IGF-1 group,tube formation increased by 60.32%(P=0.0002).7).Pathway analysis of modulation of IGF-1 expression induced by basigin-2:AKT inhibitors(LY294002)and MEK inhibitors(PD98549)were independently used to block the related pathway.The levels of IGF-1 m RNA in BV2 cells under different treatments were detected by q RT-PCR.Basigin-2 silence by si RNA and AKT inhibitor reversed the upward trend of IGF-1 after hypoxia.Moreover,the level of HIF-1,basigin,IGF-1,P-AKT,AKT,P-ERK and ERK in BV2 cells under different treatments were evaluated by western blot.The results at protein level showed that hypoxia could increase the expression of IGF-1,AKT inhibitor and basigin-2 slience by si RNA could inhibit the production of IGF-1.However,ERK inhibitor could not inhibit the expression of IGF-1.8).Molecular mechanism of IGF-1 promoting EC tube formation:IGF-1 receptor inhibitor was used to block the IGF-1 signal.The tube formation of RF/6A decreased by 101.33%(P=0.0001).The expression level of VEGF m RNA and protein was not siginificantly changed.However,the transcriptional level of VEGFR-2(P=0.0091)and protein level(P=0.0006)both decreased.ConclusionIn the present study,we firstly reported basigin was involved in in microglia-mediated regulation of oxygen-induced retinal angiogenesis.Data revealed significantly higher numbers of microglia accumulating near retinal angiogenic sprouts.Moreover,microglial expressed basigin was obviously elevated.In vitro,the positive effects of basigin-2 expressed by microglia cells under hypoxic treatment on the angiogenesis capcity of retinal vascular endothelial cells were confirmed.Moreover,the role of microglia played in facilitating tube formation is not necessarily dependend on direct cell-to-cell contact.Liposome coated si RNA was used as the research platform,and the effects of slience of microglial basigin-2 expression on the angiogenesis were focused.The molecular mechanism of basigin-2 increasing the secretion of IGF-1 by PI3K/AKT pathway was proposed and elucidated.In addition,our data revealed IGF-1 enhanced angiogenesis by up-regulating VEGFR-2 expression in RF/6A cells.The results extend our understanding of microglia.Furthermore,the regulation effect and mechanism of microglial expressed basigin-2 as transmitter and promoting hypoxia related retinal neovascularization were revealed.Thus,our study provided experimental basis for the treatment of retinal angiogenesis induced by hypoxia and ischemic.