RESUMO
AIMS: 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 Ins2Akita mice, EPAC-1 levels are decreased which is associated with an increase in microRNA-7-5p expression and reduced eNOS activity. CONCLUSION/INTERPRETATION: 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.
Assuntos
Permeabilidade Capilar/genética , Diabetes Mellitus Experimental/genética , Retinopatia Diabética/genética , Retinopatia Diabética/metabolismo , Endotélio Vascular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/genética , MicroRNAs/fisiologia , Óxido Nítrico Sintase Tipo III/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Regulação para Baixo/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
In this protocol, PAH is induced by combining a 60 mg/kg monocrotalin (MCT) injection with increased pulmonary blood flow through an aorto-caval shunt (MCT+Flow). The shunt is created by inserting an 18-G needle from the abdominal aorta into the adjacent caval vein. Increased pulmonary flow has been demonstrated as an essential trigger for a severe form of PAH with distinct phases of disease progression, characterized by early medial hypertrophy followed by neointimal lesions and the progressive occlusion of the small pulmonary vessels. To measure the right heart and pulmonary hemodynamics in this model, right heart catheterization is performed by inserting a rigid cannula containing a flexible ball-tip catheter via the right jugular vein into the right ventricle. The catheter is then advanced into the main and the more distal pulmonary arteries. The histopathology of the pulmonary vasculature is assessed qualitatively, by scoring the pre- and intra-acinar vessels on the degree of muscularization and the presence of a neointima, and quantitatively, by measuring the wall thickness, the wall-lumen ratios, and the occlusion score.