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makoto shirasawa, hiroto terasaki, noboru arimura, shozo sonoda, taiji sakamoto; Effect Of Tnf-alpha On Barrier Function In Polarized Rpe Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3699.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial (RPE) cells form a blood-ocular barrier and its polarized-nature is crucial for maintaining barrier functions. Nonetheless, highly polarized RPE cells have not been used for in vitro studies. Considering the frequent presence of tumor necrosis factor-a (TNF-a) in retinal disorders, it should be studied using polarized RPE cells to elucidate the real mechanism of retinal disorders. This study was conducted to examine the effects of TNF-a on barrier integrity of polarized RPE cells.
Porcine RPE cells (1x10^5 cells) were seeded on fibronectin-coated TranswellTM. The high polarization of RPE layer was confirmed both by its high trans-epithelial resistance (TER >150 ohm/cm2) and polarized secretion of vascular endothelial growth factor (ratio of lower chamber/upper chamber > 2.5 times), which were used for the following experiments. After starvation, RPE cells were incubated with/without 10 ng/ml TNF-a for 24 hour. Then TER was measured chronologically. The effect of TNF-a on TER or intracellular signal pathway proteins such as p38 MAPK, NF-kappa B, JNK, caspase was evaluated using specific inhibitors as follows: SB203580 (p38 MAPK inhibitor), CAPE (NF-kB inhibitor), SP600125 (JNK inhibitor), Z-VAD-fmk (caspase inhibitor). The localization of ZO-1 was evaluated by immunohistochemistry. Morphological evaluation was also performed by electron microscopy.
After incubation with TNF-a, TER decreased over time and reached 13.1 +/- 3.4 % (average +/- SD; p<0.01) of reduction in comparison to control at 24 hours, which was abolished by anti-TNF-a antibody (3.1 +/- 4.4 %). This reduction was inhibited by SB203580 inhibitor, but not by any other inhibitors. In control, ZO-1 was located clearly on a margin of RPE cells, but this localization became dispersed after co-incubation with TNF-a. Microvilli of RPE was flattened after TNF-a stimulation by electron-microscopy.
The previous reports showed that TNF-a alone does not impair barrier function of non-polarized RPE cells in vitro. While, in this study, RPE barrier function was significantly damaged by TNF-a alone in highly polarized RPE cells. This result indicates that even TNF-a alone may play an important role even in pathogenesis of mild inflammatory retinal disorders such as chronic macular degeneration.
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