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Invest Ophthalmol Vis Sci. 2017 Jul 1;58(9):3496-3505. doi: 10.1167/iovs.16-21091.

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

Author information

1
Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.
2
Ocular Angiogenesis Group, Departments of Ophthalmology and Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands 2Cellular Imaging Core Facility, Department of Medical Biology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands.

Abstract

Purpose:

Proinflammatory cytokines such as tumor necrosis factor (TNFα) 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 TNFα 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 TNFα or a combination of TNFα, IL1β, 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:

TNFα reduced the expression of tight and adherens junctions in BRECs. Permeability for a 376 Da molecular tracer was increased after TNFα stimulation, but not for larger tracers. We found that 3',5'-cyclic adenosine monophosphate (cAMP) stabilized the barrier properties of BRECs, and that TNFα significantly decreased intracellular cAMP levels. When BRECs were preincubated with a membrane-permeable cAMP analog, the effects of TNFα on claudin-5 expression and permeability were mitigated. The effects of TNFα on barrier function in BRECs were largely independent of the small Rho guanosine triphosphate (GTP)ases RhoA and Rac1, which is in contrast to TNFα effects on the nonbarrier endothelium. The combination of TNFα, IL1β, and VEGF increased permeability for a 70 kDa-FITC tracer, also mediated by cAMP.

Conclusions:

TNFα alone, or in combination with IL1β and VEGF, induces permeability of the BRB in vitro for differently sized molecular tracers mediated by cAMP, but independently of Rho/Rac signaling.

PMID:
28715583
DOI:
10.1167/iovs.16-21091
[Indexed for MEDLINE]

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