Role And Mechanism Of Microglia-Derived Spp1 In Promoting Pathological Retinal Neovascularization

Rationale:Pathological retinal neovascularization(RNV)is a defining feature of many ischemic ocular diseases,all of which result in irreversible vision loss seriously and affect the visual function and quality of life of patients.As the resident immune cells in retina,microglia play an important role in retinopathy.During pathological RNV,activated microglia migrate to affected areas and promote vascular formation by up-regulating phagocytic activity and producing pro-inflammatory and pro-angiogenic mediators.Moreover,we have not fully elucidated the physical roles in microglia-endothelial cell(EC)communication in RNV.In this study,using single-cell RNA sequencing,we revealed that in the mouse model of oxygen-induced retinopathy,pro-inflammatory secreted protein Spp1 was the most upregulated gene in microglia.Spp1(also known as Osteopontin)is a secreted multifunctional glyco-phosphoprotein that responds to acute and chronic inflammatory settings and signals via integrin and CD44 receptors.In central nervous system(CNS),microglia have been shown to be a major source of Spp1.However,the role and underlying regulatory mechanism of Spp1 in microglia,especially in microglia-EC communication during pathological RNV are not understood.Objective:We aim to observe the expression of Spp1 in mouse models of pathological ocular neovascularization,test its function in vitro and in vivo angiogenesis,and explore the underlying regulatory mechanisms of microglia-derived Spp1 in angiogenesis.Intervention of Spp1 in vivo to evaluate the therapeutic significance of targeted Spp1on RNV.Methods:1.Single-cell RNA sequencing detected the source and expression level of Spp1.Bioinformatic analysis preliminarily explore the underlying upstream mechanisms.Microglia were treated with hypoxia and lipopolysaccharide(LPS)in vitro to simulate hypoxia and inflammatory environment,and the expression of related molecules was detected by WB to verify the changes of the above pathways.2.We detected the souse and expression level of Spp1 in OIR using WB analysis and immunofluorescence staining showed co-labeling of Spp1 with microglia,Spp1with retinal ganglion cell(RGC),Spp1 with Müller cell or Spp1 with EC.Further clarify whether microglia are the main source of Spp1.3.The b End.3 endothelial cell line was cultured with recombinant Spp1.EC functions were analyzed by Ed U incorporation assay and wound healing assay.After adding to b End.3 and treating with hypoxic BV2 CM,while Spp1 neutralizing blocked those CM-induced effects.To evaluate whether Spp1 mediates the non-contact regulation of microglia on EC.4.RNA sequencing of BV2 CM-treated b End.3 treated with anti-Spp1.It’s functions were analyzed by q RT-PCR and WB.Explore possible downstream target genes.5.We injected intravitreal injections of modified anti-Spp1/Ig G control into the eyes of OIR mouse pups/mice to further verify the function of Spp1 in vivo.Whole retinal tissues were flat-mounted and subjected to immunofluorescence staining for neovascularization analysis.Moreover,ERG was performed to examine visual function.Results:1.Single-cell RNA sequencing revealed that the pro-inflammatory secreted protein Spp1 was the most upregulated gene in microglia in the mouse model of OIR.Results from WB revealed that at P12 and P17,OIR retinas had higher levels of Spp1protein expression than controls(P<0.01).Whole flat-mount and fluorescent staining analysis revealed that the number of Spp1 positive(Spp1~+)signals increased significantly both in the central zone(zone with avascular area at P12 and NV tufts at P17)and peripheral zone(zone with mainly close normal vessels)in OIR retina at P12and P17,respectively(P<0.0001).Moreover,Spp1~+signals were found in higher numbers around NV mass between the avascular central zone and the peripheral vascularized zone at OIR P17.2.Immunofluorescence analysis revealed that Spp1 was predominantly detected in extracellular spaces besides Iba1-possitive microglia surrounded vessels,and not fully co-expressed with microglia,retinal ganglion cells,Müller cells or EC,suggested its role as secretory protein.Immunofluorescence showed that expression of Spp1 was significantly decreased(P<0.001)and only appeared in areas with residual microglia after PLX5622 treatment.3.Analysis of differential gene expression and enrichment revealed that the changed genes were considerably enriched in the NF-κB and HIF-1 signaling pathways.We treated BV2 cells with hypoxia or Lipopolysaccharide(LPS).We found that expression of Spp1 and HIF-1αwere increased in microglia cultured under hypoxic condition and could be inhibited by pre-treatment with HIF-1αinhibitor GN44028(P<0.05).Furthermore,LPS treatment significantly increased both the expression and phosphorylation level of NF-κB p65 as well as the expression of Spp1(P<0.05),which could be inhibited by the NF-κB inhibitor BAY11-77028(P<0.05).4.We found that CM from hypoxia-treated microglia promoted EC proliferation and motility capacity,compared with DMEM or normoxia-treated CM(P<0.01).Moreover,adding an Spp1-neutralizing antibody(anti-Spp1)significantly repressed those effects(P<0.05).5.Bulk RNA-seq was performed to compare transcriptomes of b End.3 treated with hypoxia MG-CM plus anti-Spp1 or Ig G control.We found that Kit was significantly downregulated in anti-Spp1-treated group,which was further tested by q RT-PCR and WB(P<0.01).Moreover,anti-Spp1 treatment suppressed expression and phosphorylation level of Akt and m TOR(P<0.01).We simultaneously treated b End.3 with r Spp1,imatinib mesylate(a Kit inhibitor),and the combination of both.Results from WB showed that Spp1 overexpression significantly increased Kit expression as well as the expression and phosphorylation level of Akt/m TOR pathway,which could be reduced by imatinib mesylate treatment(P<0.05).6.IgG control or anti-Spp1 antibody were injected intravitreally at P12 of OIR mice,and whole retina tissues were collected at P17.Spp1 protein levels were significantly lower in anti-Spp1 antibody-injected retinas than in control groups(P<0.01).Compared with the control group,mice treated with anti-Spp1 antibody exhibited a significantly reduced area of NV tufts(P<0.0001).We also compared ERG amplitudes in eyes injected with normal control,Ig G control,or anti-Spp1antibody.It was observed that the anti-Spp1 antibody improved ERG amplitude(P<0.05),indicating a better neural response.Conclusions:Here,we revealed that Spp1 was upregulated in pathological RNV and dominantly secreted by microglia using OIR model with single-cell RNA sequencing.Furthermore,we showed that microglia-derived Spp1 promoted EC proliferation and migration via endothelial Kit/Akt/m TOR activation.Finally,we investigated that intraocular application of Spp1 neutralizing antibody could suppress RNV and improve visual function.Our results underline the pivotal role of Spp1 in linking microglia activation and pathological RNV formation in ischemic retinopathy,which paves the way for novel therapeutic interventions.