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.

Figure 1

Compound 49b requires Epac1 to decrease permeability. Panel A is a bar graph of the diffusive flux on REC grown in normal glucose (NG) or high glucose (HG) or HG + 50 nM Compound 49b or 10 μM VEGF. N = 4 for each group. *P < 0.05 versus NG, #P < 0.05 versus HG. Panel B shows the flux measurements of REC grown in normal or high glucose and treated with an Epac1 specific agonist. N = 4 for each group. *P < 0.05 versus NG, #P < 0.05 versus HG.

Figure 2

Epac1 depletion increases VEGF expression and PKCzeta activation. Retinal endothelial cells were grown in NG, HG, HG + Compound 49b, HG + scrambled siRNA (HG + Sc), HG + Compound49b + scrambled siRNA (Sc + 49b), HG + Epac1 siRNA, or HG + Compound 49b + Epac1 siRNA (Epac1 siRNA + 49b). Panel A is a control to show Epac1 knockdown. Panel B is the ratio of phosphorylated PKCzeta to total PKCzeta. Panel C is VEGF levels compared with beta actin. N = 4 for each group of cells. *P < 0.05 versus NG, #P < 0.05 versus HG, %P < 0.05 versus HG + Compound 49b. A representative blot is provided for each group.

Figure 3

ZO-1 levels are not increased by Compound 49b when Epac1 is knocked down. Panel A shows ZO-1 levels in REC grown in NG, HG, HG + Compound 49b, HG + scrambled siRNA (HG + Sc), HG + Compound49b + scrambled siRNA (Sc + 49b), HG + Epac1 siRNA, or HG + Compound 49b + Epac1 siRNA (Epac1 siRNA + 49b). N = 4 for each group of cells. *P < 0.05 versus NG, #P < 0.05 versus HG, %P < 0.05 versus HG + Compound 49b. A representative blot is provided for each group. Panel B shows immunostaining of ZO-1 in Epac1 floxed mouse retina versus cdh5/Epac1 Cre-Lox mice retina. Scale bar is 50 μm. Red staining is ZO-1 and Isolectin B4 is green. Arrows point to gaps along the vessel with no ZO-1 staining. Staining is representative of four mice in each group. Panel C provides Western blot results for ZO-1 levels in the Epac1 floxed and conditional knockout mice. N = 4, *P < 0.05 versus floxed.

Figure 4

Occludin is regulated by Epac1. Panel A shows occludin levels in REC grown in NG, HG, HG + Compound 49b, HG + scrambled siRNA (HG + Sc), HG + Compound49b + scrambled siRNA (Sc + 49b), HG + Epac1 siRNA, or HG + Compound 49b + Epac1 siRNA (Epac1 siRNA + 49b). N = 4 for each group of cells. *P < 0.05 versus NG, #P < 0.05 versus HG, %P < 0.05 versus HG + Compound 49b. A representative blot is provided for each group. Panel B shows representative immunostaining of occludin in Epac1 floxed mouse retina versus cdh5/Epac1 Cre-Lox mice retina. Red staining is occludin and Isolectin B4 is green. Arrows point to gaps along the vessel with no occludin staining. Staining is representative of four mice in each group. Panel C provides Western blot results for occludin levels in the Epac1 floxed and Cre-Lox mice. N = 4, *P < 0.05 versus floxed.