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T. Murakami, D. A. Antonetti; Rab4 Contributes to Tight Junction Trafficking and Endothelial Cell Permeability. Invest. Ophthalmol. Vis. Sci. 2008;49(13):2644.
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The tight junctions help create the blood-retinal barrier and loss of tight junctions contributes to vascular hyperpermeability and macular edema in diabetic retinopathy and other retinal vascular diseases. Recent studies have demonstrated trafficking of tight junction proteins regulates permeability. Multiple steps of endocytic trafficking require small G-proteins in the Rab family. Here we investigated whether the small GTPase Rab4 affects tight junction trafficking and vascular permeability.
Bovine retinal endothelial cells (BRECs) were grown to confluence and stepped to media containing 1% FBS and hydrocortisone. BRECs grown on coverslips were immunostained using the antibodies against occludin, claudin-5, ZO-1 and Rab4, followed by fluorescent secondary antibodies. Biochemical analysis of junctional protein translocation was performed by sucrose gradient ultracentrifugation followed by Western blotting, for BRECs and diabetic retinas. The permeability of BREC grown on transwell filters to 70kDa RITC labeled dextran was determined after transfection of Rab4 mutants using Amaxa nucleofection kit.
Rab4 was colocalized with occludin, claudin-5 and ZO-1, at the cell border. After VEGF treatment, Rab4 positive endosomes were identified that co-localized with tight junction proteins. Cell fractionation experiments demonstrated that VEGF increased occludin and claudin-5 content in buoyant fractions that also contained Rab4, and that occludin was shifted to buoyant fractions in diabetic retinas. Over-expression of wild type Rab4 and constitutively active Q67L mutant led to a significant increase in permeability, whereas the inactive S22N did not alter permeability.
These data suggest that Rab4 contributes to the regulation of tight junction trafficking and vascular permeability in retinal endothelial cells. Understanding the molecular mechanisms controlling tight junction trafficking may provide novel therapeutic targets to treat diseases of elevated vascular permeability such as diabetic retinopathy.
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