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  • Title: Different Effects of Thrombin on VEGF Secretion, Proliferation, and Permeability in Polarized and Non-polarized Retinal Pigment Epithelial Cells.
    Author: Terasaki H, Shirasawa M, Otsuka H, Yamashita T, Uchino E, Hisatomi T, Sonoda S, Sakamoto T.
    Journal: Curr Eye Res; 2015 Sep; 40(9):936-45. PubMed ID: 25310246.
    Abstract:
    We investigated the effect of thrombin on the secretion of vascular endothelial growth factor (VEGF), on cellular proliferation, and on the integrity of the barrier function of polarized retinal pigment epithelial (RPE) cells. In addition, we compared the responses of polarized to that of non-polarized RPE cells. Porcine polarized RPE cells were established using Transwell membranes. The polarization of the RPE cells was determined by their high transepithelial electrical resistance (TER > 200 Ω cm(2)) and by their differential secretion of VEGF (basal direction >apical direction by 2.5×). RPE cells were incubated with thrombin (5-20 U/ml) for 24 h. The concentration of VEGF in the culture medium was measured by enzyme-linked immunosorbent assay, and the TER was measured. Cellular proliferation was assessed by Ki-67 immunostaining. The area of laser-induced choroidal naovascularization (CNV) was measured in rat eyes and compare to that of controls with or without thrombin. Our results showed that thrombin significantly increased VEGF secretion both in polarized and non-polarized RPE cells in a dose-dependent way. Thrombin did not significantly affect the TER or the expression of tight-junctional proteins in polarized RPE cells, but decreased it in non-polarized RPE cells by inducing intercellular gaps. Ki-67-positive cells were observed in non-polarized RPE cells but not in polarized RPE cells as controls. After thrombin exposure, the number of Ki-67-positive cells increased significantly in non-polarized RPE cells but not in polarized RPE cells. The area of CNV was larger in thrombin-injected eye than control eyes. Although thrombin increased VEGF secretion regardless of cell polarity, its effects on proliferation and barrier integrity were dependent upon cell polarity. Cell polarization is an important factor for determining the response of RPE cells to thrombin, and the different responsive patterns to thrombin upon cell polarity might explain the complicated pathology of such diseases as age-related macular degeneration.
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