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Carla Jhoana Ramos, Xuwen Liu, Chengmao Lin, David Antonetti; Rap1 activation promotes vascular blood-retinal barrier. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4048.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Activation of Rap1 through the cAMP-dependent guanine nucleotide exchange factor EPAC was reported to enhance barrier function in human umbilical endothelial cells. However, the effect of EPAC-Rap1 signaling on retinal endothelial barrier, tight junctions, and the relationship to vascular endothelial growth factor (VEGF)-induced permeability are unknown. We hypothesize that EPAC-Rap1 activation inhibits VEGF-induced permeability in retinal endothelial cells.
Methods: Primary, bovine retinal endothelial cells (BREC) were used to model the blood retinal barrier. The EPAC-specific activator cAMP analog 8-pCPT-2-O-Me-cAMP-AM (8CPT) was used. Cell monolayer permeability was measured by electrical resistance using the ECIS-ZΘ system or by measuring 70kDa RITC dextran flux across transwell filters. Rap1 activation was determined using a Rap1 capture assay with GST-RalGDS followed by Western blot. EPAC specific antagonist (ESI-09) or Rap1B siRNA (bovine) were used to inhibit EPAC or silence Rap1 expression in BREC. Confocal microscopy assessed junctional protein organization. Male Sprague-Dawley rats were used to test retinal vascular permeability in vivo.
Results: Treatment of BREC monolayers with 8CPT and activation of Rap1 prevented both VEGF or VEGF and tumor necrosis factor (TNF)-induced permeability to 70kDa dextran (p<0.0001) and reduction in electrical resistance (p<0.0001). 8CPT treatment reversed VEGF-induced permeability to solute and ion flux. EPAC inhibition or Rap1 silencing led to an increase in basal permeability and VEGF had no additional permeability increase. Pre-treatment with 8CPT decreased VEGF signaling in the Erk pathway but Rap1 knockdown revealed no effect on VEGF signaling. Immunofluorescence staining showed disruption of tight junction organization in VEGF treated cells and 8CPT blocked VEGF junctional disruption (p<0.0001). Permeability studies in vivo showed that VEGF/TNF increased permeability and 8CPT blocked VEGF/TNF permeability.
Conclusions: These data demonstrate that activation of Rap1 through EPAC in retinal endothelial cells promotes barrier properties, inhibits and reverses VEGF and VEGF/TNF-induced permeability, and protects tight junction organization. Collectively, these data suggest activation of Rap1 through cAMP analogs may provide a therapeutic means to restore barrier properties in diseases of increased retinal permeability.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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