Abstract
BackgroundPrevious 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).MethodsThe 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).ResultsMMP-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.ConclusionsThe 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.
Highlights
Choroidal neovascularization (CNV) occurs in wet-type age-related macular degeneration, extreme myopia, and pathological myopia [1,2,3]
Increased CNV and matrix metallopeptidase-2 (MMP-2) staining in the choroid-retinal pigment epithelial (RPE) membrane of the CNV model Krypton laser photocoagulation was used to create CNV in the right eyes of six to eight week-old mice; the left eyes of the mice were used as controls
The RPE layer was destroyed and CD31 staining was present in the subretinal space in the CNV group, indicating that laser treatment destroyed the choroid-RPE barrier at the laser spots and induced endothelial cells to migrate to the subretinal space, which is evidence that successful CNV was created (Figure 1 top row, right panel)
Summary
Choroidal neovascularization (CNV) occurs in wet-type age-related macular degeneration, extreme myopia, and pathological myopia [1,2,3]. Angiogenesis in CNV is a complicated process with imbalance in the production of pro-angiogenic and anti-angiogenic factors (for example, excessive production of vascular endothelial growth factor (VEGF)), leading to pathological neovascularization [4]. The process of angiogenesis as it occurs in CNV involves the proteolysis of an extracellular matrix, the proliferation and migration of endothelial cells, and the synthesis of a new matrix in the destination [5]. Matrix metalloproteinase (MMP) family proteins play important roles in angiogenesis partly due to their ability to degrade basal membrane and extracellular matrix proteins. The activity of MMP is down-regulated by endogenous inhibitors, namely tissue inhibitors of metalloproteinase [7]. Previous reports have indicated that matrix metallopeptidase-2 (MMP-2) regulates angiogenic processes, which are involved in choroidal neovascularization (CNV). 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)
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