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Hiroto Terasaki, Makoto Shirasawa, Hiroki Otsuka, Shozo Sonoda, Taiji Sakamoto; Effects of thrombin on tight junction-associated molecules in polarized RPE cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):6069.
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© ARVO (1962-2015); The Authors (2016-present)
Retinal pigment epithelial cells (RPEs) is highly polarized epithelial cells and function as a barrier between the retina and choroid. Although the previous studies showed thrombin exerts many effects on RPEs, most of them used non-polarized RPEs, which might not necessarily reflect an in vivo condition. We reported thrombin upregulates VEGF secretion in polarized RPEs (ARVO, 2012). In this study, we investigated effects of thrombin on tight junction-associated molecules including ZO-1 and F-actin in polarized and non-polarized RPEs.
Porcine polarized RPE was established in Boyden chamber by our previous report (Sonoda S, et al. Nature Protoc, 2009). Polarized or non-polarized RPEs were treated with thrombin (4U/ml). Expression of ZO-1 or F-actin was observed by imunofuorescence.
In control, ZO-1 was strongly expressed at cell border in polarized RPEs but weakly expressed in non-polarized RPEs. Although F-actin was also observed at cell border in polarized RPEs, it was not at cell border but in cytosol in non-polarized RPEs. After thrombin stimulation in polarized RPEs, expression of ZO-1 and F-actin was not affected. Even when concentration of thrombin was increased (10-20U/ml), same results were obtained. On the contrary, thrombin decreased expression of ZO-1 and made expression of F-actin diffused in non-polarized RPEs.
Expression pattern of tight junction associated molecules and effects of thrombin differ dependent upon cell polarity. Polarized RPE might be more suitable than for non-polarized RPEs for analyzing the pathophysiology of RPE in vivo.
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