title: The microRNA-7-mediated reduction in EPAC-1 contributes to vascular endothelial permeability and eNOS uncoupling in murine experimental retinopathy.
abstract: OBJECTIVE
  To investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy.
  
  
  METHODS
  Oxygen-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.
  
  
  RESULTS
  Hypoxia 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 Ins2Akitamice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity.
  
  
  CONCLUSIONS
  In 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.
authors.affiliation: Group of Research in Pharmacology of Chronic Diseases (CDPHARMA), Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University of Heidelberg, Heidelberg, Germany.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University Medical Center Groningen, Groningen, The Netherlands.|Cardiovascular Regenerative Medicine (CAVAREM), Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1 (EA11), 9713 GZ, Groningen, The Netherlands.
authors.forename: Veronica
authors.initials: V
authors.lastname: Garcia-Morales
authors.affiliation: International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University of Heidelberg, Heidelberg, Germany.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University Medical Center Groningen, Groningen, The Netherlands.|Cardiovascular Regenerative Medicine (CAVAREM), Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1 (EA11), 9713 GZ, Groningen, The Netherlands.|Section of Endocrinology, 5th Medical Department, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Julian
authors.initials: J
authors.lastname: Friedrich
authors.affiliation: Cardiovascular Regenerative Medicine (CAVAREM), Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1 (EA11), 9713 GZ, Groningen, The Netherlands.
authors.forename: Lysanne M
authors.initials: LM
authors.lastname: Jorna
authors.affiliation: Group of Research in Pharmacology of Chronic Diseases (CDPHARMA), Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), University of Santiago de Compostela, 15782, Santiago de Compostela, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Campos-Toimil
authors.affiliation: International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University of Heidelberg, Heidelberg, Germany.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University Medical Center Groningen, Groningen, The Netherlands.|Section of Endocrinology, 5th Medical Department, Medical Faculty Mannheim, University of Heidelberg, Heidelberg, Germany.
authors.forename: Hans-Peter
authors.initials: HP
authors.lastname: Hammes
authors.affiliation: International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University of Heidelberg, Heidelberg, Germany.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University Medical Center Groningen, Groningen, The Netherlands.|Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands.
authors.forename: Martina
authors.initials: M
authors.lastname: Schmidt
authors.affiliation: International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University of Heidelberg, Heidelberg, Germany. g.krenning@umcg.nl.|International Research and Training Network on Diabetic Microvascular Complications (GRK1874/DIAMICOM), University Medical Center Groningen, Groningen, The Netherlands. g.krenning@umcg.nl.|Cardiovascular Regenerative Medicine (CAVAREM), Department of Pathology and Medical Biology, University Medical Center Groningen, University of Groningen, Hanzeplein 1 (EA11), 9713 GZ, Groningen, The Netherlands. g.krenning@umcg.nl.
authors.forename: Guido
authors.identifier: http://orcid.org/0000-0001-5850-5667
authors.initials: G
authors.lastname: Krenning
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D002199:Capillary Permeability / Q000235:genetics*; D002478:Cells, Cultured; D003921:Diabetes Mellitus, Experimental / Q000150:complications / Q000235:genetics* / Q000378:metabolism; D003930:Diabetic Retinopathy / Q000235:genetics* / Q000378:metabolism*; D015536:Down-Regulation / Q000235:genetics; D004730:Endothelium, Vascular / Q000378:metabolism*; D020662:Guanine Nucleotide Exchange Factors / Q000235:genetics*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D035683:MicroRNAs / Q000502:physiology*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*
chemical_list: C482601:Epac protein, mouse; D020662:Guanine Nucleotide Exchange Factors; C549243:MIRN7 microRNA, mouse; D035683:MicroRNAs; D052250:Nitric Oxide Synthase Type III; C496315:Nos3 protein, mouse
keywords: EPAC-1; Endothelial cell; Hypoxia; MicroRNA-7; Retinopathy