Garcia-Morales Veronica, Friedrich Julian, Jorna Lysanne M, Campos-Toimil Manuel, Hammes Hans-Peter, Schmidt Martina, Krenning Guido
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.
Acta Diabetol. 2017 Jun;54(6):581-591. doi: 10.1007/s00592-017-0985-y. Epub 2017 Mar 28.
To investigate the consequences of oxidative stress and hypoxia on EPAC-1 expression during retinopathy.
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.
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 Ins2 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.
研究氧化应激和缺氧对视网膜病变期间EPAC-1表达的影响。
在小鼠中诱导氧诱导性视网膜病变，并通过免疫荧光研究EPAC-1表达。利用计算机分析确定内皮细胞中EPAC-1表达与微小RNA-7-5p之间的联系，并通过对表达微小RNA-7-5p的细胞进行蛋白质免疫印迹分析加以证实。在体外，将内皮细胞置于2%氧气环境中孵育或转染微小RNA-7-5p，评估这些处理对EPAC-1表达、内皮细胞高通透性和一氧化氮（NO）生成的影响。在Ins2Akita小鼠模型中，通过定量聚合酶链反应（qPCR）评估EPAC-1表达水平以及微小RNA-7-5p水平。在Ins2Akita模型中通过免疫印迹法评估内皮型一氧化氮合酶。
缺氧诱导微小RNA-7-5p表达，其通过翻译抑制作用抑制内皮细胞中EPAC-1的表达，导致高通透性和内皮型一氧化氮合酶（eNOS）活性丧失。环磷酸腺苷（cAMP）类似物8-pCPT-2'-O-Me-cAMP对EPAC-1的激活降低了EPAC-1对氧化应激的敏感性，并将内皮通透性恢复至基线水平。此外，8-pCPT-2'-O-Me-cAMP挽救了eNOS活性和NO生成。在视网膜病变小鼠模型，即氧诱导性视网膜病变和自发性糖尿病杂合Ins2小鼠中，EPAC-1水平降低，这与微小RNA-7-5p表达增加和eNOS活性降低有关。
结论/解读：在视网膜病变中，EPAC-1表达以微小RNA-7介导的方式降低，导致内皮功能障碍。残余EPAC-1的药理学激活可挽救内皮功能。总体而言，这些数据表明EPAC-1似乎是改善（糖尿病）视网膜病变的有效且可成药的靶分子。
