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B. E. Phillips, J. Tarbell, D. A. Antonetti; Occludin Regulates Paracellular Permeability and Cell Division in ARPE19 Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2513.
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The Blood Retinal Barrier restricts the movement of proteins, ions, and fluid into the retina and is established by well-developed tight junctions in the vascular endothelial and retinal pigmented epithelial cells (RPE). Disregulation of tight junction permeability is characteristic of various pathologies including ischemia and diabetic retinopathy. The content of the tight junction protein occludin is closely correlated with barrier properties. Although occludin ablation and over expression studies have provided insight, little is known regarding the role of occludin in control of epithelial permeability under physiologic conditions. We hypothesize that reduction of occludin content will increase RPE permeability under physiologic pressure.
Occludin protein content was reduced using siRNA in ARPE19 cells. RPE monolayers were grown on transwell filters, and paracellular permeability of 70kDa rhodamine-isothiocyanate conjugated dextran (RITC-dextran) and 467Da tetramethylrhodamine (TAMRA) were measured with 10cm water pressure or diffusively with no water pressure. Cell proliferation was measured by tritiated thymidine incorporation and staining for the cell cycle marker Ki67.
Occludin content reduction increased diffusive permeability to TAMRA by 15% and 50% with 10cm of water pressure applied. The larger 70kd RITC-dextran displayed no change in diffusive permeability and a 50% decrease in permeability with 10cm of water pressure. Trans-electrical resistance was decreased 50% with occludin content reduction indicating increased ion permeability. Cellular division rates were increased by 70% after occludin content reduction. Cell cycle inhibitors demonstrated that the increased rate of cell division did not affect paracellular permeability.
These data suggests that occludin impacts paracellular routes of transport that differentiate the 70kDa dextran and 467Da TAMRA molecules. This data supports previous work demonstrating changes in permeability under diffusive conditions and reveals that occludin contributes to the cell’s adaptive response to pressure. Further this data shows for the first time that occludin effects rates of cell division.
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