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C. A. Aveleira, E. B. Wolpert, A. F. Ambrosio, D. A. Antonetti; TNF Signals Through NFB to Induce Retinal Endothelial Cell Permeability. Invest. Ophthalmol. Vis. Sci. 2009;50(13):21.
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Diabetic retinopathy has characteristics of a low-grade chronic inflammatory disease. Diabetes increases interleukin-1β (IL-1β) and tumor necrosis factor-alpha (TNF) content in the vitreous of diabetic patients and in diabetic rat retinas commensurate with elevated retinal vascular permeability. In this study, we investigated the mechanisms by which TNF increases retinal endothelial cell permeability. Further, the ability of PKCζ/NFΚB inhibition to prevent cytokine-induced permeability was evaluated.
Primary cultures of bovine retinal endothelial cells (BREC) were grown on transwell filters and exposed to TNF. Confluent BREC were pretreated with dexamethasone, for 18 h, or chemical inhibitors of PKC isoforms or IKK, for 30 min, and then stimulated with TNF for 6 h. BREC permeability to 70 kDa RITC-dextran was measured. Western blotting and immunocytochemistry for tight junction proteins were performed to determine their content and localization.
TNF increases retinal endothelial cell permeability. TNF treatment decreases ZO-1 and claudin-5 content and disrupts tight junction organization at cell border. The TNF effect on cell permeability and tight junction proteins was completely blocked by dexamethasone. IKK inhibitor partially prevented TNF-induced retinal endothelial cell permeability. Atypical PKC, but not conventional PKC inhibition, completely prevented TNF-induced retinal endothelial cell permeability. Finally, the changes in the tight junction protein content and localization induced by TNF treatment were blocked by atypical PKC inhibition.
These results demonstrate that TNF alters the tight junction complex and increases retinal endothelial cell permeability. Dexamethasone treatment completely prevents the TNF-induced increase in retinal endothelial cell permeability. Furthermore, TNF signals to tight junction disruption and endothelial cell permeability requires PKCζ and NFΚB activation. Together, these data suggest that targeting atypical PKC may provide a specific therapeutic target for the prevention of inflammatory cytokine-induced vascular permeability
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