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Tami Livnat, Omer Bialer, Yael Nisgav, Rima Dardik, Dov Weinberger; Evaluation Of The Effect Of Thrombin On The Structure Of Tight Junctions And The Permeability Of RPE And Endothelial Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):930.
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The blood retinal barriers (BRB) integrity is essential for normal retinal function. Any damage to the BRB integrity is accompanied by infiltration and edema, which may lead to vision loss. BRB integrity is maintained by a series of tight junction proteins. The tight junction system is a dynamic system capable of regulating the intercellular space, thus controlling the passage of substances of varying size: high density of tight junction proteins ensures tight intercellular contact and reducing barrier permeability. Damage to the BRB and vision loss accompanied by elevated thrombin levels was found in some of the retinal pathological conditions, such as diabetic retinopathy, inflammation, vascular occlusions, surgical complications and tumors. Understanding the effects of thrombin on BRB integrity is of great importance. The aim of the study was to explore the effects of thrombin on the permeability of endothelial and retinal pigment epithelial (RPE) cells due to alterations in the expression and distribution of tight junction proteins.
We used an endothelial cell line and a retinal pigment epithelium cell line. The expression of genes encoding tight junction proteins (claudin5, Jam1, 2 and ZO1) was evaluated using real time PCR. Permeability was evaluated based on spectrophotometric monitoring of the leakage of labeled dextran molecules of varying size. Intracellular localization of tight junction proteins was studied using immunohistochemistry staining.
In both endothelial and RPE cell lines, addition of thrombin induced elevation in cell permeability as indicated by increased dextran leakage through endothelial and RPE monolayers. The effect of thrombin was dose dependent and the elevation in permeability occurred in a few minutes after thrombin addition to the cells. The expression of genes of the tight junction family was altered in both cell lines. In endothelial cells, thrombin induced downregulation of JAM1 expression, and upregulation of claudin 5 expression. ZO1 and JAM2 mRNA levels were not affected by thrombin. At the protein level, changes in claudin 5 were demonstrated in immunohistochemical staining.
The data indicate that elevation in thrombin level may cause alterations in the blood barriers of the retina through changes in endothelial and RPE permeability. Changes in the expression and localization of tight junction proteins induced by thrombin may partially contribute to increased permeability.
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