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Carla Jhoana Ramos, David Antonetti; Activation of EPAC1-Rap1 Prevents and Reverses VEGF Induced Endothelial Permeability . Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2270.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Activation of Rap1 through the cAMP-dependent guanine nucleotide exchange factor EPAC1 was reported to enhance barrier function in human umbilical endothelial cells through rasip1 regulation of Rho activation. However, the role of EPAC1 in retinal endothelial barrier and the relationship to vascular endothelial growth factor (VEGF) induced permeability is unknown. We hypothesize that EPAC1 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 cAMP analog 8-pCPT-2-O-Me-cAMP-AM (8CPT) was used to specifically activate EPAC1. Cell monolayer permeability was measured by electrical resistance using the ECIS-ZΘ system or by measuring 70kDa RITC dextran flux across transwell filters. Rap1A activation was determined using a Rap capture assay with GST-RalGDS to capture active Rap1A followed by Western blot. RhoA activity was measured using Rho GTP binding protein ELISA assay.
Results: Western blot of BREC lysates revealed the presence of EPAC1, Rap1A, and rasip1. Treatment of BREC monolayers with VEGF increased permeability to 70kDa dextran (p<0.0001) and reduced electrical resistance (p<0.0001). Pre-treatment of BREC with 8CPT completely prevented both the VEGF-induced permeability to dextran (p<0.0001) and the reduction in electrical resistance (p<0.0001). Importantly, treatment of BREC with VEGF followed by 8CPT treatment reversed VEGF-induced permeability to solute and ion flux. Compared to control and VEGF treatments, 8CPT and 8CPT+VEGF treatments revealed an increase in Rap1A activation (p<0.001) as well as an increase in rasip1 and Rap1A co-immunoprecipitation. Immunocytochemistry revealed that tight junctional proteins ZO-1, occludin, and claudin-5 were disrupted in cells treated with VEGF and protected in cells treated with 8CPT prior to VEGF treatment.
Conclusions: These data demonstrate that activation of Rap1A in retinal endothelial cells through the cAMP dependent guanine nucleotide exchange factor EPAC1 promotes barrier properties and inhibits VEGF-induced permeability. Importantly, Rap1 activation reversed VEGF induced permeability. Collectively, these data suggest activation of Rap1 may provide a therapeutic means to restore barrier properties in diseases of increased retinal permeability.
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