Ye Eun-Ah, Liu Li, Steinle Jena J
Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI, USA.
Department of Anatomy and Cell Biology, Wayne State University, Detroit, MI, USA; Department of Ophthalmology, Wayne State University, Detroit, MI, USA.
Vision Res. 2017 Oct;139:23-29. doi: 10.1016/j.visres.2017.07.007. Epub 2017 Aug 7.
Hyperglycemia is a significant risk factor for diabetic retinopathy and induces multiple biochemical changes, including inflammation and endothelial dysfunction in the retina. Alterations in microRNA expression have been implicated in the pathological responses of diabetic retinopathy and the manipulation of microRNA may provide powerful strategy for therapeutics. Among the predicted targets of miR-15a and -16 are TGF-beta3, SMAD2/3, and VEGF, all of which are known to play a role in vascular endothelial functions. The purpose of this study was to investigate the hypothesis that miR-15a/16 inhibits TGF-beta3/VEGF signaling to maintain retinal endothelial cell barrier protein levels. Human primary retinal endothelial cells (REC) were maintained in normal (5mM) glucose or transferred to high glucose medium (25mM) for 3days. REC were transfected with miRNA mimics (hsa-miR-15a-5p and -16-5p). Retinal lysates from miR-15a-transgenic mice were also analyzed. We demonstrated that overexpression of miR-15a/16 resulted in decreased TGF-beta3 signaling and VEGF levels in cultured REC grown in high glucose conditions. In addition, the levels of tight junction proteins, zonula occludens-1 (ZO-1) and occludin, were elevated in REC following overexpression of miR-15a and -16. Overexpression of miR-15a and -16 played a role in reducing cellular permeability through inhibition of VEGF signaling in REC cultured under high glucose conditions. Using miR-15a-transgenic mice, we demonstrated the regulatory role of miR-15a on TGF-beta3 signaling and tight junction proteins in vivo. Our outcomes suggest that miR-15a/16 maintain the retinal endothelial cell barrier by reducing TGFbeta3/VEGF signaling and increasing levels of key tight junction proteins.
高血糖是糖尿病视网膜病变的一个重要危险因素，可引发多种生化变化，包括视网膜中的炎症和内皮功能障碍。微小RNA表达的改变与糖尿病视网膜病变的病理反应有关，对微小RNA的调控可能为治疗提供有力策略。miR-15a和-16的预测靶标包括TGF-β3、SMAD2/3和VEGF，所有这些都已知在血管内皮功能中发挥作用。本研究的目的是探讨miR-15a/16抑制TGF-β3/VEGF信号传导以维持视网膜内皮细胞屏障蛋白水平的假说。人原代视网膜内皮细胞（REC）在正常（5mM）葡萄糖中培养，或转移至高糖培养基（25mM）中培养3天。REC用miRNA模拟物（hsa-miR-15a-5p和-16-5p）转染。还分析了来自miR-15a转基因小鼠的视网膜裂解物。我们证明，在高糖条件下培养的REC中，miR-15a/16的过表达导致TGF-β3信号传导和VEGF水平降低。此外，miR-15a和-16过表达后，REC中紧密连接蛋白小带闭合蛋白-1（ZO-1）和闭合蛋白的水平升高。miR-15a和-16的过表达通过抑制高糖条件下培养的REC中的VEGF信号传导，在降低细胞通透性方面发挥作用。使用miR-15a转基因小鼠，我们在体内证明了miR-15a对TGF-β3信号传导和紧密连接蛋白的调节作用。我们的结果表明，miR-15a/16通过降低TGFβ3/VEGF信号传导和增加关键紧密连接蛋白的水平来维持视网膜内皮细胞屏障。
