Rationale:Microglia with a repertoire of functions are critical in pathological regulation of angiogenesis in the retina. However, retinal microglia with beneficial contributions and corresponding mechanisms during pathological neovascularization are poorly understood.Methods:We conducted a bioinformatic comparison of public single-cell RNA transcriptome data between retinal microglia from mice with oxygen-induced retinopathy (OIR) and an antiangiogenic microglial population named MG3 from the spine. The essential beneficial factor thrombospondin-1 (Tsp-1) from microglia was discovered and then validated in the retina of mice with OIR at P17. Exosomes were isolated from Tsp-1-knockout microglia (KO-Exos) and Tsp-1+microglia (NT-Exos). Human umbilical vein endothelial cells (HUVEC) morphology studies, exosomes' miRNA sequencing, luciferase reporter assay, miRNA loss of function studies, and intravitreal injection were used to explore the mechanism of Tsp-1 and microglia-associated retinal angiogenesis.Results:The bioinformatic analyses of single-cell RNA-seq data indicated that a subtype of retinal microglia named RMG1 shares features with MG3 in regulating wound healing, cell adhesion, and angiogenesis. Remarkably, Tsp-1, an extracellular matrix protein with robust inhibition of angiogenesis, was especially expressed in both MG3 and RMG1. However, the scarcity of Tsp-1+cells was observed in RMG1, which could be an obstacle to attenuating retinal neovascularization. Subsequently, we found that exosomes derived from Tsp-1+microglia inhibit the migration and tube formation of HUVEC. Moreover, the knockout of Tsp-1 led to the enrichment of miR-27a-5p in exosomes from microglia and promoted angiogenesis compared to that of NT-Exosin vitro. Furthermore, in the luciferase reporter assay on the transcriptional activity of the promoter, we demonstrated that Tsp-1 negatively regulates miR-27a-5p expression. In addition, SMAD family member 3 (Smad3), a receptor-activated Smad protein that is conducive to vascular homeostasis, was defined as a functional target gene of miR-27a-5p. These data were consistently confirmedin vivoin the retina of mice with OIR.Conclusion:Collectively, the Tsp-1/miR-27a-5p/Smad3 axis is involved in microglia-related and exosome-mediated antiangiogenic regulation of the retina. Therefore, this study reveals a novel mechanism by which retinal microglia maintain vascular homeostasis, thereby providing a new therapeutic target for pathological neovascularization.