Artikel
Identification of myeloid cell populations at sites of laser-induced choroidal neovascularization by single-cell profiling
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Autoren
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Anja Schlecht
- Freiburg/D
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P. Wieghofer
- Institute of Neuropathology, University of Freiburg/D; Institute of Anatomy, University Leipzig/D
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P. Zhang
- Freiburg/D
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S. Bonvea
- Freiburg/D
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M. Gruber
- Freiburg/D
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R. Sankowski
- Institute of Neuropathology, University of Freiburg/D
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G. Schlunck
- Freiburg/D
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H. Agostini
- Freiburg/D
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M. Prinz
- Institute of Neuropathology, University of Freiburg/D
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C. Lange
- Freiburg/D
| Veröffentlicht: | 5. Februar 2020 |
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Gliederung
Text
Background: Myeloid cells (MC), such as resident microglia (MG) and infiltrating blood-derived monocytes (MO), are key players in the formation of choroidal neovascularization (CNV). However, the specific discrimination between MG and MO is challenging and the precise function of MG during CNV development remains unclear. In this study, we used MG-specific reporter mice to perform single-cell RNA-Sequencing (scRNA-Seq) of MG cells with and without laser-induced CNV to analyze their transcriptional profile.
Methods: Adult Cx3cr1CreER/+:Rosa26-Tomfl/+ mice were used for RNA-Seq analysis. Six laser spots were applied to each eye by an Argon laser (532nm). Laser setting were 150 mW, 100 ms with a spot size of 100 µm to induce CNV formation, while untreated littermates served as controls. Retinal MG cells were sorted by FACS on the third day after laser injury to perform scRNA-Seq. Protein expression of most outstanding factors was investigated by ELISA and immunohistochemistry. As a therapeutic approach, antibodies against SPP1 were injected intravitreally one day after laser injury and CNV lesion size was quantified.
Results: ScRNA-Seq analysis demonstrates, that MG strongly change their expression profile following laser-induced CNV formation and that different MG cell populations are present at sites of laser-induced CNV. In the CNV-associated MG cluster GO terms involved in migration, immune response and DNA synthesis were enriched and genes associated with proliferation, hypoxia-sensing and angiogenesis were significantly upregulated. SPP1, also known as Osteopontin, was highly expressed in CNV-associated MG. Immunohistochemistry and ELISA protein analysis reveals that SPP1 accumulates in the choroid and intravitreal injection of anti-SPP1 antibodies lead to an increased lesion size when compared to control treated eyes.
Conclusion: Our study demonstrates that retinal MG cells strongly change their expression profile following laser-induced CNV formation. CNV-associated MG cells secrete a plethora of molecules among which SPP1 may have anti-angiogenic and thus neuroprotective properties.
