Electron microscopic immunocytochemical demonstration of blood-retinal barrier breakdown in human diabetics and its association with aldose reductase in retinal vascular endothelium and retinal pigment epithelium

Summary Light-microscopic immunohistochemical staining for albumin has been used to localize sites of blood-retinal barrier (BRB) breakdown in ocular disorders, but the mechanism for BRB compromise cannot be resolved at this level. Using eyes up to 2 days post-mortem from normal patients or from patients with diabetic retinopathy, or other disorders known to cause BRB failure, electron-microscopic immunocytochemistry reveals focal breakdown of the inner BRB, comprised of the retinal vascular endothelium (RVE), which appears to be mediated by diffuse permeation of the RVE cells and by vesicular transport. Permeation of the retinal pigmented epithelial (RPE) cells that comprise the outer BRB also occurs, but there is no evidence of opening of tight junctions between RVE or RPE in any of the disorders evaluated. Increased aldose reductase (AR) expression in the RVE and RPE cells of diabetics as well as in the perivascular retinal astrocytes, which interact with RVE cells to establish the inner BRB, suggests that AR activity and the subsequent intracellular accumulation of sorbitol in these cell types may impair the function of the BRB in diabetes.     

Comparative subcellular localization of NRF2 and KEAP1 during the hepatocellular carcinoma development in vivo

The activation of Nuclear Factor, Erythroid 2 Like 2 – Kelch Like ECH Associated Protein 1 (NRF2-KEAP1) signaling pathway plays a critical dual role by either protecting or promoting the carcinogenesis process. However, its activation or nuclear translocation during hepatocellular carcinoma (HCC) progression has not been addressed yet. This study characterizes the subcellular localization of both NRF2 and KEAP1 during diethylnitrosamine-induced hepatocarcinogenesis in the rat. NRF2-KEAP1 pathway was continuously activated along with the increased expression of its target genes, namely Nqo1, Hmox1, Gclc, and Ptgr1. Similarly, the nuclear translocation of NRF2, MAF, and KEAP1 increased in HCC cells from weeks 12 to 22 during HCC progression. Likewise, colocalization of NRF2 with KEAP1 was higher in the cell nuclei of HCC neoplastic nodules than in surrounding cells. Moreover, immunofluorescence analyses revealed that the interaction of KEAP1 with filamentous Actin was disrupted in HCC cells. This disruption may be contributing to the release and nuclear translocation of NRF2 since the cortical actin cytoskeleton serves as anchoring of KEAP1. In conclusion, this evidence indicates that NRF2 is progressively activated and promotes the progression of experimental HCC.     

KEAP1基因及其突变位点对非小细胞肺癌细胞株作用的实验初步研究

摘要 目的：研究KEAP1基因及KEAP1基因突变位点对肺癌细胞株的作用。方法：通过Western blot 方法,比较携带KEAP1 基因突变的肺癌细胞株（A549,NCI-H460,NCI-H838）与KEAP1 基因野生型的肺癌细胞株（NCI-H292, NCI-H1299, 95D, SPC-A1）之间,NRF2基因与NRF2下游基因HO-1的蛋白表达水平,检测并比较两组细胞的活性氧（ROS）含量;以新发现的非小细胞肺癌（NSCLC）病人的 KEAP1 体细胞突变作为模板构建 KEAP1 突变质粒,对自身存在 KEAP1 突变的肺癌细胞株A549,通过改造的 pMSCV 逆病毒转染体系,分别构建过表达空载,野生型及突变型 KEAP1 的 A549 稳定细胞株,比较过表达不同质粒的细胞间丙二醛（MDA）含量及 NRF2 下游抗氧化等相关基因的表达水平;通过克隆形成实验检测细胞增殖情况。结果：KEAP1基因突变组肺癌细胞株与KEAP1基因无突变的对照组细胞株相比,NRF2和HO-1蛋白表达显著增高,活性氧水平显著降低（P<0.01）;过表达野生型KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因转录水平表达显著下降（P<0.01）,蛋白水平表达下降,细胞内丙二醛水平显著增高（P<0.01）,克隆形成率显著降低（P<0.01）,而过表达突变型 KEAP1 与过表达空载的 A549细胞相比,NRF2 及其下游基因表达、细胞内丙二醛水平、克隆形成率均无显著差异（P>0.05）。结论：KEAP1基因具有抑癌作用,其突变为失活型突变,突变后KEAP1/NRF2通路激活,KEAP1基因突变可能通过改变细胞的氧化应激水平,促进肺癌的发生发展。     

Lycium barbarum polysaccharides reduce neuronal damage, blood-retinal barrier disruption and oxidative stress in retinal ischemia/reperfusion injury

Neuronal cell death, glial cell activation, retinal swelling and oxidative injury are complications in retinal ischemia/reperfusion (I/R) injuries. Lycium barbarum polysaccharides (LBP), extracts from the wolfberries, are good for "eye health" according to Chinese medicine. The aim of our present study is to explore the use of LBP in retinal I/R injury. Retinal I/R injury was induced by surgical occlusion of the internal carotid artery. Prior to induction of ischemia, mice were treated orally with either vehicle (PBS) or LBP (1 mg/kg) once a day for 1 week. Paraffin-embedded retinal sections were prepared. Viable cells were counted; apoptosis was assessed using TUNEL assay. Expression levels of glial fibrillary acidic protein (GFAP), aquaporin-4 (AQP4), poly(ADP-ribose) (PAR) and nitrotyrosine (NT) were investigated by immunohistochemistry. The integrity of blood-retinal barrier (BRB) was examined by IgG extravasations. Apoptosis and decreased viable cell count were found in the ganglion cell layer (GCL) and the inner nuclear layer (INL) of the vehicle-treated I/R retina. Additionally, increased retinal thickness, GFAP activation, AQP4 up-regulation, IgG extravasations and PAR expression levels were observed in the vehicle-treated I/R retina. Many of these changes were diminished or abolished in the LBP-treated I/R retina. Pre-treatment with LBP for 1 week effectively protected the retina from neuronal death, apoptosis, glial cell activation, aquaporin water channel up-regulation, disruption of BRB and oxidative stress. The present study suggests that LBP may have a neuroprotective role to play in ocular diseases for which I/R is a feature.     

CircRNA expression profile and functional analysis in retinal ischemia-reperfusion injury

Retinal ischemia-reperfusion (I/R) is involved in the pathogenesis of many vision-threatening diseases. circRNAs act as key players in gene regulation and human diseases. However, the global circRNA expression profile in retinal I/R injury has not been fully uncovered. Herein, we established a murine model of retinal I/R injury and performed circRNA microarrays to identify I/R-related circRNAs. 1265 differentially expressed circRNAs were identified between I/R retinas and normal retinas. Notably, the detection of cWDR37 level in aqueous humor could discriminate glaucoma patients from cataract patients (AUC = 0.9367). cWdr37 silencing protected against hypoxic stress- or oxidative stress-induced retinal ganglion cell (RGC) injury. cWdr37 silencing alleviated IR-induced retinal neurodegeneration as shown by increased NeuN staining, reduced retinal reactive gliosis, and decreased retinal apoptosis. Collectively, this study provides a novel insight into the pathogenesis of retinal I/R injury. cWdr37 is a promising target for the diagnosis or treatment of I/R-related ocular diseases.