Compound 49b Regulates ZO-1 and Occludin Levels in Human Retinal Endothelial Cells and in Mouse Retinal Vasculature

Abstract

Purpose

To 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.

Methods

Primary 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.

Results

High 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.

Conclusions

Compound 49b increased ZO-1 and occludin protein levels, likely leading to decreased permeability.