The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy

Veronica Garcia-Morales,Guido Krenning,Manuel Campos-Toimil,Julian Friedrich,Hans-Peter Hammes,Lysanne M Jorna,Martina Schmidt

doi:10.1007/s00592-017-0985-y

Abstract

AimsTo investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy.MethodsOxygen-induced retinopathy was induced in mice and EPAC-1 expression investigated by immunofluorescence. In silico analyses were used to identify a link between EPAC-1 expression and microRNA-7-5p in endothelial cells and confirmed by western blot analyses on cells expressing microRNA-7-5p. In vitro, endothelial cells were either incubated at 2% oxygen or transfected with microRNA-7-5p, and the effects of these treatments on EPAC-1 expression, endothelial hyperpermeability and NO production were assessed. In the Ins2Akita mouse model, levels of EPAC-1 expression as well as microRNA-7-5p were assessed by qPCR. Endothelial nitric oxide synthase was assessed by immunoblotting in the Ins2Akita model.ResultsHypoxia induces the expression of microRNA-7-5p that translationally inhibits the expression of EPAC-1 in endothelial cells, resulting in hyperpermeability and the loss of eNOS activity. Activation of EPAC-1 by the cAMP analogue 8-pCPT-2′-O-Me-cAMP reduced the sensitivity of EPAC-1 to oxidative stress and restored the endothelial permeability to baseline levels. Additionally, 8-pCPT-2′-O-Me-cAMP rescued eNOS activity and NO production. In mouse models of retinopathy, i.e., oxygen-induced retinopathy and the spontaneous diabetic heterozygous Ins2Akita mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity.Conclusion/InterpretationIn retinopathy, EPAC-1 expression is decreased in a microRNA-7-mediated manner, contributing to endothelial dysfunction. Pharmacological activation of remnant EPAC-1 rescues endothelial function. Collectively, these data indicate that EPAC-1 resembles an efficacious and druggable target molecule for the amelioration of (diabetic) retinopathy.

Highlights

Introduction reduction in exchange protein activated by Cyclic adenosine monophosphate (cAMP) (EPAC)1 expression is associated with the hypoxia-induced expression of microRNA-7, resulting in translational repression
The effect of hypoxia on EPAC-1 expression was confirmed in endothelial cell cultures exposed to 2% or 20% oxygen, where EPAC-1 protein expression decreased (2.4-fold, p \ 0.01, Fig. 1d, e) when cells were exposed to hypoxia for 24 h
We show that hypoxia induces the expression of miR7 by endothelial cells in vitro and in vivo, reducing the expression of EPAC-1

Summary

Results

Oxygen-induced retinopathy is associated with a marked decrease in EPAC-1 expression (Fig. 1a). Co-transfection of COS7 cells with miR-7 mimics and the EPAC-1 reporter construct decreased luciferase activity (2.6-fold) compared to scrambled controls (p \ 0.05), whereas the luciferase activity of the EPAC-2 reporter was unaffected (Fig. 2c). These data indicate that EPAC-1 is a specific target of miR7. Treating miR-7-expressing cells with 8-pCPT reduced endothelial hyperpermeability (1.7-fold, p \ 0.05), Fig. 2 Hypoxia induces microRNA-7-mediated suppression of EPAC-1. The addition of miR-7 mimics to endothelial cells did imitate the hypoxia-induced loss of eNOS activity as indicated by a reduction in eNOS phosphorylation at serine 1177 (p-eNOS/eNOS ratio; 1.7-fold decrease, p \ 0.01; Fig. 4e). Table 4) and found no other transcript which was decreased in both conditions

Discussion

Compliance with ethical standards