Dataset Information

Roles of NF?B-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization.

ABSTRACT: BACKGROUND: Previous reports have indicated that matrix metallopeptidase-2 (MMP-2) regulates angiogenic processes, which are involved in choroidal neovascularization (CNV). However, the regulation of MMP-2 in CNV has not been well-characterized. To gain more information about the regulation of MMP-2 in CNV, we analyzed the circuitry associated with MMP-2 regulation in a CNV model and in cell cultures, focusing on NF?B and the microRNA-29 family (miR-29s). METHODS: The CNV model was established by subjecting C57BL/6 mice to fundus photocoagulation with a krypton red laser. In choroidal-retinal pigment epithelial (RPE) tissues of the model, immunohistochemistry was used to evaluate the angiogenesis and MMP-2 expression; reverse-transcription quantitative PCR (RT-qPCR) was used to determine the levels of miR-29s; and western blot was used to analyze the protein levels of nuclear factor kappa-light-chain-enhancer of activated B cells (NF?B) inhibitor, I?B?, and its phosphorylated form, phospho-I?B?. At the cellular level, RT-qPCR was used to examine the levels of miR-29s following NF?B activation by tumor necrosis factor alpha (TNF?); and western blot and luciferase assay were used to determine the regulation of MMP-2 by miR-29s in a human RPE cell line (ARPE-19) and in an umbilical vein endothelial cell line (EA hy926). RESULTS: MMP-2 staining was increased in the choroidal neovascular membrane of laser-treated retina. Also, the NF?B pathway was induced in choroid-RPE tissue, as evidenced by a lower protein level of I?B? and a higher level of phospho-I?B? in the tissue homogenates than in those from non-treated eyes. During the period when the NF?B pathway was induced, reduced miR-29s were detected in the choroidal-RPE tissue of the laser-treated eyes. In cultured ARPE-19 cells, TNF? decreased miR-29a, b, and c, and the effects were rescued by NF?B decoy. In ARPE-19 and EA hy926, miR-29s mimics reduced the contents of secreted MMP-2 in the culture media. We also documented that miR-29s reduced MMP-2 3'-UTR-mediated luciferase transcription. CONCLUSIONS: The results suggest that in CNV, NF?B activation inhibits miR-29s, which may contribute to angiogenesis by up-regulating the MMP-2 protein level in RPE cells. These observations may help in developing a strategy for resolving CNV by targeting miR-29s levels.

SUBMITTER: Cai J

PROVIDER: S-EPMC4030271 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Roles of NFκB-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization.

Cai Jingjing J Yin Guibin G Lin Bing B Wang Xianwei X Liu Xiaoling X Chen Xiaoyan X Yan Dongsheng D Shan Ge G Qu Jia J Wu Shengzhou S

Journal of neuroinflammation 20140515

<h4>Background</h4>Previous reports have indicated that matrix metallopeptidase-2 (MMP-2) regulates angiogenic processes, which are involved in choroidal neovascularization (CNV). However, the regulation of MMP-2 in CNV has not been well-characterized. To gain more information about the regulation of MMP-2 in CNV, we analyzed the circuitry associated with MMP-2 regulation in a CNV model and in cell cultures, focusing on NFκB and the microRNA-29 family (miR-29s).<h4>Methods</h4>The CNV model was ...[more]

PMID: 24886609

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Project description:BackgroundNeovascular age-related macular degeneration (nAMD) commonly causes vision loss from aberrant angiogenesis, termed choroidal neovascularization (CNV). Macrophages are heterogeneous cells that are necessary for experimental CNV, present in human CNV samples, and can display diverse functions, which are dependent upon both their origin and tissue microenvironment. Despite these associations, choroidal macrophage heterogeneity remains unexplored.MethodsWe performed multi-parameter flow cytometry on wildtype (WT) and Ccr2-/- mice after laser injury to identify macrophage subtypes, and determine which subsets originate from classical monocytes. To fate map tissue resident macrophages at steady state and after laser injury, we used the Cx3cr1CreER/+ ; Rosa26zsGFP/+ mouse model. We reanalyzed previously published single-cell RNA-seq of human choroid samples from healthy and nAMD patients to investigate human macrophage heterogeneity, disease association, and function.ResultsWe identified 4 macrophage subsets in mice: microglia, MHCII+CD11c-, MHCII+CD11c+, and MHCII-. Microglia are tissue resident macrophages at steady state and unaffected by laser injury. At steady state, MHCII- macrophages are long lived, tissue resident macrophages, while MHCII+CD11c- and MHCII+CD11c+ macrophages are partially replenished from blood monocytes. After laser injury, MHCII+CD11c- macrophages are entirely derived from classical monocytes, MHCII- macrophages originate from classical monocytes (90%) and an expansion of tissue resident macrophages (10%), and MHCII+CD11c+ macrophages are derived from classical monocytes (70%), non-classical monocytes (10%), and an expansion of tissue resident macrophages (20%). Single-cell RNA-seq analysis of human choroid found 5 macrophage subsets: two MHCII+CD11C- and three MHCII+CD11C+ populations. One MHCII+CD11C+ subset was 78% derived from a patient with nAMD. Differential expression analysis identified up-regulation of pro-angiogenic gene expression in one MHCII+CD11C- and two MHCII+CD11C+ subsets, including the disease-associated cluster. The upregulated MHCII+CD11C- pro-angiogenic genes were unique compared to the increased MHCII+CD11C+ angiogenesis genes.ConclusionsMacrophage origin impacts heterogeneity at steady state and after laser injury in mice. Both mice and human patients demonstrate similar macrophage subtypes. Two discrete pro-angiogenic macrophage populations exist in the human choroid. Targeting specific, pro-angiogenic macrophage subsets is a potential novel therapeutic for nAMD.

Dataset Information

Roles of NF?B-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization.

Publications

Roles of NFκB-miR-29s-MMP-2 circuitry in experimental choroidal neovascularization.

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