Abstract
PurposeTo investigate whether Epac1 is key to Compound 49b's regulation of zonula occluden 1 (ZO-1) and occludin levels in human retinal endothelial cells (REC) and in an Epac1 vascular-specific conditional knockout mouse retina.MethodsPrimary REC were grown in normal (5 mM) or high glucose (25 mM). Some cells were treated with a novel β-adrenergic receptor agonist, Compound 49b. Additional dishes were treated with Epac1 siRNA or Compound 49b + Epac1 siRNA. Protein levels of ZO-1, occludin, VEGF, and protein kinase C zeta (PKCz) were measured by Western blotting. Cell permeability was measured in REC grown in normal or high glucose and treated with Compound 49b, a specific Epac 1 agonist (8-CPT-2′-O-Me-cAMP), or VEGF. Epac1 floxed and cdh5-Cre mice were bred to generate Epac1 knockout mice in vascular endothelial cells. Immunofluorescence was done on retinal flatmounts from the floxed and Cre-Lox mice for occludin and ZO-1. Western blotting was also done for both proteins in whole retinal lysates from the mice.ResultsHigh glucose significantly reduced ZO-1 and occludin protein levels, which was associated with reduced cell adhesion. Compound 49b increased endothelial cell barrier protein levels through active Epac1. Knockout of Epac1 in vascular endothelial cells substantially reduced ZO-1 and occludin staining in retinal flatmounts, as well as protein levels.ConclusionsCompound 49b increased ZO-1 and occludin protein levels, likely leading to decreased permeability.
Highlights
To investigate whether Epac[1] is key to Compound 49b’s regulation of zonula occluden 1 (ZO-1) and occludin levels in human retinal endothelial cells (REC) and in an Epac[1] vascular-specific conditional knockout mouse retina
High glucose significantly reduced zonula occludens (ZO)-1 and occludin protein levels, which was associated with reduced cell adhesion
Knockout of Epac[1] in vascular endothelial cells substantially reduced ZO-1 and occludin staining in retinal flatmounts, as well as protein levels
Summary
We have already shown that Compound 49b could reduce VEGF, as well as activate PKA, the goal of this study was to investigate whether active Epac was required for Compound 49b to regulate REC barrier proteins
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.