Jalimarada Supriya S, Ogando Diego G, Bonanno Joseph A
School of Optometry, Indiana University, Bloomington, IN.
Mol Vis. 2014 Dec 12;20:1668-79. eCollection 2014.
Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A., is a major cause of corneal transplantation. FECD is associated with mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8, and LOXHD1. The current pathological description of the dystrophy includes deficiency of corneal endothelium (CE) pump function and induction of the unfolded protein response (UPR). This study aims to determine the contribution of the two mechanisms by assessing the expression levels of (1) seven endothelial ion transporters known to regulate stromal hydration and (2) UPR related genes in a set of six CE samples obtained from FECD patients compared to that of normal controls.
CE samples collected during FECD keratoplasty or from an eye bank (normal control) were transferred into an RNA stabilizing agent and refrigerated. Total RNA from each CE specimen was individually extracted. The expression levels of ion transporters and UPR genes were tested using quantitative real-time (RT) PCR and a UPR specific PCR array, respectively.
In normal CE, the comparative expression levels of ion transporters in decreasing order were SLC4A11, Na(+)/K(+) ATPase, pNBCe1, and NHE1, followed by the isoforms of monocarboxylate transporters (MCTs). In FECD samples, Na(+)/K(+) ATPase and MCTs 1 and 4 were significantly downregulated compared to normal controls (p<0.05). The PCR array tested 84 UPR related genes. Data analysis showed upregulation of 39 genes and downregulation of three genes, i.e., approximately 51% of the tested genes had their expression altered in FECD samples with a difference greater than ± twofold regulation. Thirteen of the altered genes showed significant changes (p<0.05). The PCR array results were validated by quantitative RT-PCR.
FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes. The significant downregulation of ion transporters indicates simultaneous compromised CE pump function in Fuchs' dystrophy.
在美国，富克斯角膜内皮营养不良（FECD）影响约5%的40岁以上人群，是角膜移植的主要原因。FECD与多种不相关基因的突变有关：SLC4A11、COL8A2、TCF8和LOXHD1。目前对该营养不良的病理学描述包括角膜内皮（CE）泵功能缺陷和未折叠蛋白反应（UPR）的诱导。本研究旨在通过评估（1）七种已知调节基质水合作用的内皮离子转运蛋白和（2）与正常对照相比，从FECD患者获得的一组六个CE样本中的UPR相关基因的表达水平，来确定这两种机制的作用。
在FECD角膜移植手术期间或从眼库收集的CE样本（正常对照）被转移到RNA稳定剂中并冷藏。分别从每个CE标本中提取总RNA。分别使用定量实时（RT）PCR和UPR特异性PCR阵列检测离子转运蛋白和UPR基因的表达水平。
在正常CE中，离子转运蛋白的相对表达水平从高到低依次为SLC4A11、Na(+)/K(+)ATP酶、pNBCe1和NHE1，其次是单羧酸转运蛋白（MCTs）的同工型。在FECD样本中，与正常对照相比，Na(+)/K(+)ATP酶以及MCTs 1和4显著下调（p<0.05）。PCR阵列检测了84个UPR相关基因。数据分析显示39个基因上调，3个基因下调，即约51%的检测基因在FECD样本中的表达发生改变，差异大于±两倍调节。其中13个改变的基因显示出显著变化（p<0.05）。PCR阵列结果通过定量RT-PCR验证。
FECD样本有明显的UPR，蛋白质加工途径基因的表达有显著变化。离子转运蛋白的显著下调表明在富克斯营养不良中CE泵功能同时受损。
