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

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 kappa 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 kappa B) inhibitor, I kappa B alpha, and its phosphorylated form, phospho-I kappa Ba. At the cellular level, RT-qPCR was used to examine the levels of miR-29s following NF kappa B activation by tumor necrosis factor alpha (TNF alpha); 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 kappa B pathway was induced in choroid-RPE tissue, as evidenced by a lower protein level of I kappa Ba and a higher level of phospho-I.Ba in the tissue homogenates than in those from non-treated eyes. During the period when the NF kappa B pathway was induced, reduced miR-29s were detected in the choroidal-RPE tissue of the laser-treated eyes. In cultured ARPE-19 cells, TNFa decreased miR-29a, b, and c, and the effects were rescued by NF kappa 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 kappa 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.