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Brett Poulis, Aikun Wang, Jing Cui, Luba Kojic, Joanne Matsubara; TNF-α and Aβ promote activation of metalloproteinases in retinal pigment epithelial cells and is mediated by the NF-κB pathway: implications for AMD pathogenesis. Invest. Ophthalmol. Vis. Sci. 2013;54(15):147.
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Inflammation and oxidative stress play important roles in the pathogenesis of age-related macular degeneration (AMD). Unfortunately, the interactions between these processes have yet to be elucidated. Drusen components, such as Aβ, may trigger pro-inflammatory events in the outer retina via activation of the NF-κB pathway - causing injury to retinal pigment epithelial (RPE) cells, increased cytokine production, and matrix metalloproteinase (MMP) activation. Inflammation-induced MMP activation causes extracellular matrix (ECM) degradation and Bruch's membrane malfunction. To investigate the role of inflammation and drusen on the activation of MMPs in RPE cells, we carried out a series of experiments to study the link between TNF-α or Aβ induced MMP activation and the activation of NF-κB.
Long Evans rats (N=6) were intravitreously injected with a 5 μL solution containing 0.2 ng of TNF-α, 7 μg of Aβ(1-40), or 7 μg of Aβ(40-1). Eyes were enucleated after 24 hours, cryosections were prepared, and in-situ gel zymography using DQ-Gelatin (LifeTechnologies Inc.) was used to detect MMP activity. Immunofluorescence was imaged with a confocal microscope (Zeiss) at 40x. In vitro studies were undertaken with an ARPE19/ NF-κB stable cell line with luciferase as the reporter protein. Reporter cells were seeded and treated with Aβ(1-40) or Aβ(40-1) at a concentration of 0.1, 0.3, or 1 μM. Recombinant hTNF-α at a concentration of 20 ng/mL for 8 hrs was also carried out. All experiments were performed in quadruplicate.
Intravitreous injections of TNF-α and Aβ resulted in RPE MMP activation as demonstrated by in-situ gel zymography. Compared with untreated reporter cells, recombinant hTNF-α significantly up-regulated NF-κB activity up to 12.64 fold, indicating a robust reporter system was established. No differences were seen in NF-κB activity using stimulation dosages of Aβ(1-40) at 0.1 and 0.3 μM, but NF-κB was 1.31 fold higher at 1 μM.
Intravitreous injections with TNF-α or Aβ resulted with RPE MMP activation. A drusen component, Aβ, up-regulated NF-κB activity. Further assessment of MMP activation by drusen components is needed to gain further insight into the pathological changes in RPE, possibly leading to strategies that can limit abnormal MMP activation associated with AMD pathogenesis.
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