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Tami Livnat, Omer Bialer, Yael Nisgav, Mor Dachbash, Rima Dardik, Dov Weinberger; Short-term exposure to thrombin induces long lasting disruption of barrier properties and proangiogenic signals in RPE. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3655.
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Thrombin is a multifunctional protease playing a central role in coagulation. Apart from its major function in vein and artery occlusion, thrombin was found to play a significant role in inflammatory diseases. Thrombin exerts cellular effects through its membrane receptor (PAR). In vivo testing of thrombin is difficult since its existence in plasma is short-term and transient. The retinal pigment epithelium (RPE) forms the outer blood retina barrier (BRB) and its integrity is essential for normal function of the retina. Retinal pathologies involving BRB disruption, such as diabetic retinopathy, inflammation, vascular occlusions and tumors may be accompanied by elevation in thrombin levels. In the present work, we aimed to explore the impact of thrombin on the integrity and function of the RPE blood barrier. We induced in-vitro a short-term exposure of RPE to pathological levels of thrombin and studied the immediate and long lasting changes in the RPE permeability and angiogenic balance.
ARPE-19 cells were grown for a month to achieve definite polarity properties. Following a short (10 minutes) exposure to thrombin, the cells were washed and covered with new medium until measurements were performed. Permeability was evaluated based on spectrophotometric monitoring of the leakage of labeled dextran molecules. The expression of pro- and anti- angiogenic genes was evaluated using real time PCR. Protein levels were measured by ELISA or by FlowCytomix™. MMPs activity was examined by zymography.
Short-term exposure to thrombin induced a long lasting (for hours) increase in RPE permeability to 10, 40 and 70 KD dextran. Decrease in PEDF mRNA and increase in VEGF and HGF mRNA expression and protein levels were detected even 24 hours after the 10 minute exposure to thrombin. MMPs 2 and 9 activities were also increased hours after the short-term exposure to thrombin.
Short-term exposure to thrombin induces proangiogenic signals in RPE and disruption of barrier properties. The data indicate that changes in permeability, gene expression, proteins levels and activity persisted for hours after short-term exposure to thrombin. Based on our findings, we suggest that accumulation of short-term exposures to thrombin over years contributes to the pathological processes involved in CNV development in the elderly.
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