title: Roles of NFκB-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 NFκB 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 (NFκB) inhibitor, IκBα, and its phosphorylated form, phospho-IκBα. At the cellular level, RT-qPCR was used to examine the levels of miR-29s following NFκB activation by tumor necrosis factor alpha (TNFα); 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 NFκB pathway was induced in choroid-RPE tissue, as evidenced by a lower protein level of IκBα and a higher level of phospho-IκBα in the tissue homogenates than in those from non-treated eyes. During the period when the NFκB pathway was induced, reduced miR-29s were detected in the choroidal-RPE tissue of the laser-treated eyes. In cultured ARPE-19 cells, TNFα decreased miR-29a, b, and c, and the effects were rescued by NFκB 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, NFκB 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.
authors.forename: Jingjing
authors.initials: J
authors.lastname: Cai
authors.forename: Guibin
authors.initials: G
authors.lastname: Yin
authors.forename: Bing
authors.initials: B
authors.lastname: Lin
authors.forename: Xianwei
authors.initials: X
authors.lastname: Wang
authors.forename: Xiaoling
authors.initials: X
authors.lastname: Liu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Chen
authors.forename: Dongsheng
authors.initials: D
authors.lastname: Yan
authors.forename: Ge
authors.initials: G
authors.lastname: Shan
authors.forename: Jia
authors.initials: J
authors.lastname: Qu
authors.affiliation: School of Optometry and Ophthalmology and Eye Hospital, Wenzhou Medical University, 270 Xueyuan Road, Wenzhou, Zhejiang 325003, People's Republic of China. shzhwu1@gmail.com.
authors.forename: Shengzhou
authors.initials: S
authors.lastname: Wu
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D002461:Cell Line, Transformed; D020256:Choroidal Neovascularization / Q000209:etiology / Q000378:metabolism* / Q000473:pathology*; D004195:Disease Models, Animal; D005123:Eye / Q000473:pathology; D005786:Gene Expression Regulation / Q000528:radiation effects; D006801:Humans; D008028:Light Coagulation / Q000009:adverse effects; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000378:metabolism*; D016328:NF-kappa B / Q000378:metabolism*; D013997:Time Factors; D014162:Transfection
chemical_list: D015703:Antigens, CD; C547014:MIRN29 microRNA, mouse; D035683:MicroRNAs; D016328:NF-kappa B; D020778:Matrix Metalloproteinase 2