TNFα-Induced Disruption of the Blood-Retinal Barrier In Vitro Is Regulated by Intracellular 3',5'-Cyclic Adenosine Monophosphate Levels.

PURPOSE. Proinflammatory cytokines such as tumor necrosis factor ( TNFa) may have a causative role in blood-retinal barrier (BRB) disruption, which is an essential step in the development of diabetic macular edema. The purpose of our study was to determine whether TNFa increases permeability in an in vitro model of the BRB and to explore the mechanisms involved. METHODS. Primary bovine retinal endothelial cells (BRECs) were grown on Transwell inserts and cells were stimulated with TNF alpha or a combination of TNF alpha, IL1 beta, and VEGF. Molecular barrier integrity of the BRB was determined by gene and protein expression of BRB-specific components, and barrier function was assessed using permeability assays. RESULTS. TNFa reduced the expression of tight and adherens junctions in BRECs. Permeability for a 376 Da molecular tracer was increased after TNF alpha stimulation, but not for larger tracers. We found that 3',5'-cyclic adenosine monophosphate (cAMP) stabilized the barrier properties of BRECs, and that TNF alpha significantly decreased intracellular cAMP levels. When BRECs were preincubated with a membrane-permeable cAMP analog, the effects of TNF alpha on claudin-5 expression and permeability were mitigated. The effects of TNF alpha on barrier function in BRECs were largely independent of the small Rho guanosine triphosphate (GTP) ases RhoA and Rac1, which is in contrast to TNF alpha effects on the nonbarrier endothelium. The combination of TNF alpha, IL1 beta, and VEGF increased permeability for a 70 kDa-FITC tracer, also mediated by cAMP. CONCLUSIONS. TNFa alone, or in combination with IL1b and VEGF, induces permeability of the BRB in vitro for differently sized molecular tracers mediated by cAMP, but independently of Rho/ Rac signaling