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Activated microglia induce the production of reactive oxygen species and promote apoptosis of co-cultured retinal microvascular pericytes

  • Basic Science
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Abstract

Purpose

Pericyte apoptosis is a predominant feature of early diabetic retinopathy. In diabetic retinopathy, activated microglia migrate and release proinflammatory cytokines that contribute to disruption of the blood–retinal barrier, neuronal loss, and enhanced ROS production. Reactive oxygen species (ROS) are implicated in pericyte death; however, the mechanism by which activated microglia affect retinal microvascular pericytes is unclear. We hypothesized that activated microglia may promote pericyte apoptosis by enhancing ROS production.

Methods

Lipopolysaccharide (LPS)-activated microglia and pericytes were co-cultured in a cell culture system. Pericyte ROS production and the mitochondrial membrane potential (ΔΨm) were determined by flow cytometry. The pericyte protein expression levels of NADPH oxidase subunits, uncoupling protein 2, nuclear NF-κB-p65, and caspase-3 were determined by western blotting. One-way ANOVAs were used for statistical analysis.

Results

LPS successfully activated the microglia, as demonstrated by their morphological and phenotype changes and the significant increase in tumor necrosis factor secretion (P < 0.01). Co-culture with activated microglia significantly up-regulated NADPH oxidase subunits (NOX4, NOX2, and NCF1; P < 0.01) and down-regulated uncoupling protein 2 expression (P < 0.01) in pericytes. Pericyte ROS production increased by 20% in the activated microglia co-cultured group, and was inhibited by pretreatment with diphenyleneiodonium, coenzyme Q10, and N-acetylcysteine. The proapoptotic pericyte changes induced by co-culture with activated microglia included a 9.50% decrease in pericyte ΔΨm and significant increases in NF-κB-p65 nuclear translocation (P < 0.01) and activated caspase-3 (P < 0.01). These proapoptotic effects of activated microglia were inhibited by diphenyleneiodonium.

Conclusions

Our results are consistent with our hypothesis that activated microglia may promote pericyte apoptosis by enhancing ROS production. Further studies are needed to examine retinal microglia activation and the corresponding changes in pericytes in a rat model of diabetes mellitus.

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Acknowledgements

Haixiang Wu conceived and designed the experiments; Xinyi Ding performed the experiments with the assistance of Meng Zhang and Ruiping Gu. Xinyi Ding analyzed the data and prepared the manuscript. Haixiang Wu is the guarantor of this study and, as such, had full access to all the data used, and takes responsibility for the integrity of the data and the accuracy of the analyses.

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Authors and Affiliations

  1. Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai, 200031, People’s Republic of China

    Xinyi Ding, Meng Zhang, Ruiping Gu, Gezhi Xu & Haixiang Wu

  2. Institute of Eye Research, Eye and ENT Hospital of Fudan University, Shanghai, People’s Republic of China

    Xinyi Ding, Meng Zhang, Ruiping Gu, Gezhi Xu & Haixiang Wu

  3. Institute of Brain Science, Fudan University, Shanghai, People’s Republic of China

    Gezhi Xu

  4. State Key Laboratory of Medical Neurobiology, Fudan University, Shanghai, People’s Republic of China

    Gezhi Xu

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Correspondence to Haixiang Wu.

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Funding

This work was supported by funding from the National Natural Science Foundation for Young Scholars of China (nos. 81300781 and 81400410). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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All authors certify that they have no affiliations with or involvement in any organization or entity with any financial interest (such as honoraria; educational grants; participation in speakers’ bureaus; membership, employment, consultancies, stock ownership, or other equity interest; and expert testimony or patent-licensing arrangements) or non-financial interest (such as personal or professional relationships, affiliations, knowledge or beliefs) in the subject matter or materials discussed in this manuscript.

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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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Ding, X., Zhang, M., Gu, R. et al. Activated microglia induce the production of reactive oxygen species and promote apoptosis of co-cultured retinal microvascular pericytes. Graefes Arch Clin Exp Ophthalmol 255, 777–788 (2017). https://doi.org/10.1007/s00417-016-3578-5

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