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Alexa K. Klettner, Stefan Koinzer, Tim Meyer, Johann B. Roider; Activation Of TLR-3 Does Not Reduce VEGF Secretion But Induces Concentration Dependent Cell Death In Porcine RPE. Invest. Ophthalmol. Vis. Sci. 2011;52(14):3009.
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The activation of TLR-3 receptor has recently been described to protect from experimentally induced choroidal neovascularisation in mice. A proposed mechanism was the reduction of Vascular Endothelial Growth Factor (VEGF) by non-targeted dsRNA. In this study, we investigated the effect of TLR-3 activation on porcine cultivated retinal pigment epithelium (RPE) cells.
For the experiments, primary porcine RPE cells of second passage were used. The expression of TLR-3 on porcine RPE cells was detected with flow cytometry. TLR-3 activation was induced by polyinolinic acid:polycytidylic acid (Poly I:C). Cell viability was investigated with trypan blue exclusion assay and cell death was detected with a cell death detection ELISA. The secretion of IFN-ß, an indicator of TLR-3 activation, and VEGF was assessed in corresponding ELISAs according to the manufacture's instruction. Mitogen-activated protein kinases (MAPK) JNK, p38 and ERK1/2 were inhibited with commercially available inhibitors. The activation of ERK1/2 was examined in Western blot with phospho-specific antibodies.
Porcine RPE expressed TLR-3 receptor. Poly I:C induced the secretion of IFN-ß. Poly I:C induced concentration-dependent cell death, detected in trypan blue exclusion assay and cell death ELISA. JNK inhibition significantly reduced cell death, while ERK 1/2 inhibition enhanced cell death slightly but significantly. ERK1/2 was activated by TLR-3 stimulation. Activation of TLR-3 induced a slight, concentration-dependent increase in VEGF secretion, which was not mediated by MAPK.
The induction of cell death in RPE cells and the increase of VEGF secretion by TLR-3 stimulation indicate that TLR-3 activation does not protect against choroidal neovascularisation by attenuation of VEGF secretion. Its toxicity on RPE cells also casts doubt about a general beneficial effect of this pathway. On the contrary, it might play a role in areolar atrophy of the RPE.
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