Single-cell transcriptome analyses reveal microglia types associated with proliferative retinopathy
Zhiping Liu, … , Ruth B. Caldwell, Yuqing Huo
Zhiping Liu, … , Ruth B. Caldwell, Yuqing Huo
Published October 20, 2022
Citation Information: JCI Insight. 2022;7(23):e160940. https://doi.org/10.1172/jci.insight.160940.
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Research Article Angiogenesis Ophthalmology

Single-cell transcriptome analyses reveal microglia types associated with proliferative retinopathy

Abstract

Pathological angiogenesis is a major cause of irreversible blindness in individuals of all age groups with proliferative retinopathy (PR). Mononuclear phagocytes (MPs) within neovascular areas contribute to aberrant retinal angiogenesis. Due to their cellular heterogeneity, defining the roles of MP subsets in PR onset and progression has been challenging. Here, we aimed to investigate the heterogeneity of microglia associated with neovascularization and to characterize the transcriptional profiles and metabolic pathways of proangiogenic microglia in a mouse model of oxygen-induced PR (OIR). Using transcriptional single-cell sorting, we comprehensively mapped all microglia populations in retinas of room air (RA) and OIR mice. We have unveiled several unique types of PR-associated microglia (PRAM) and identified markers, signaling pathways, and regulons associated with these cells. Among these microglia subpopulations, we found a highly proliferative microglia subset with high self-renewal capacity and a hypermetabolic microglia subset that expresses high levels of activating microglia markers, glycolytic enzymes, and proangiogenic Igf1. IHC staining shows that these PRAM were spatially located within or around neovascular tufts. These unique types of microglia have the potential to promote retinal angiogenesis, which may have important implications for future treatment of PR and other pathological ocular angiogenesis–related diseases.

Authors

Zhiping Liu, Huidong Shi, Jiean Xu, Qiuhua Yang, Qian Ma, Xiaoxiao Mao, Zhimin Xu, Yaqi Zhou, Qingen Da, Yongfeng Cai, David J.R. Fulton, Zheng Dong, Akrit Sodhi, Ruth B. Caldwell, Yuqing Huo

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Figure 6

The unique PRAM are localized near the pathological neovascular tufts.

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The unique PRAM are localized near the pathological neovascular tufts. (...
(A) Representative immunofluorescence images of PKM2 (green), microglia (IBa1, pink), vessel (isolectin B4, red), and nuclei (DAPI, blue) in whole-mount retinas of RA and OIR mice at P17. Scale bar: 50 μm (first, second, and fourth rows) and 10 μm (third and fifth rows). NC, negative controls; stained with isotype IgG of PKM2 and IBa1 antibody. The fluorescence intensity of PKM2 staining was calculated by ImageJ software (NIH) (5 images × 3 regions were obtained from n = 5 retinas of each group). Data are mean ± SEM. ***P < 0.001 versus RA by nonparametric Mann–Whitney U test. (B) FABP5 immunofluorescence staining of retinas from RA and OIR mice at P17. Representative FABP5 (green), CD11b (pink), isolectin B4 (red), DAPI (nuclei, blue), and merged images captured with confocal fluorescence microscopy. Scale bar: 50 μm (first, second, and fourth rows) and 10 μm (third and fifth rows). Stained with isotype IgG of FABP5 and CD11b antibody. The fluorescence intensity of FABP5 staining was calculated by ImageJ software (5 images × 3 regions were obtained from n = 5 retinas of each group). Data are mean ± SEM. ***P < 0.001 versus RA by nonparametric Mann–Whitney U test. (C) Representative immunofluorescence images of nuclear Ki67 (green), myeloid cells (CD11b, red), and vessel (isolectin B4, blue) in whole-mount retinas of RA and OIR mice at P17 (n = 4 per group). Scale bar: 50 μm and 20 μm (far right). (D) Immunofluorescence imaging of nuclear Ki67+ (white asterisk labelled, green), microglia/macrophages (F4/80, pink), and vessel (isolectin B4, blue) in whole-mount retinas of RA and OIR mice at P17 (n = 4 per group). Scale bar: 50 μm and 20 μm (far right).