Identification of Circulating Fibrocytes and Dendritic Derivatives in Corneal Endothelium of Patients with Fuchs' Dystrophy [RT-qPCR array CAPH10410]. Homo sapiens

PURPOSE: Fuchs’ endothelial corneal dystrophy (FECD) is a degenerative eye disorder affecting 4% of Americans older than 40. It is the leading indication for corneal endothelial (CE) transplantation for which there is a global donor shortage. This study aimed to gain further insight into the pathophysiology of FECD and identify targets for nonsurgical therapy. METHODS: CE from patients with late-onset FECD was compared with that of normal controls using microarray expression analysis (n = 4 FECD, n = 4 normal), reverse transcriptase quantitative PCR (n = 9 FECD, n = 8 normal), and immunohistology (n = 55 FECD, n = 15 normal). RESULTS: This led to the identification of circulating fibrocytes and their dendritic derivatives in all examined CE samples with FECD (in all clinical stages of symptomatic FECD and independent of prior cataract surgery). These cells were not present in normal CE. In this study we characterize their morphology, protein expression profile, number, and localization within the CE layer of patients with FECD. CONCLUSIONS: Circulating fibrocytes and their dendritic derivatives are a new aspect of FECD that deserves further investigation. Because they are known to cause fibrosis in a variety of organs, they may play a similar role in FECD and might be a valuable target for nonsurgical therapy. Overall design: This study was performed on CE monolayers with Descemet membrane from patients with late-onset FECD. Microarray expression analysis was performed on 8 samples (n = 4 FECD, n = 4 normal). RT-qPCR was done for 179 genes of interest and at least three reference genes (n = 9 FECD, n = 8 normal). Molecules of interest were validated with immunohistochemical and immunofluorescent staining on fresh corneal endothelial whole-mounts (n = 55 FECD, n = 15 normal). All stainings were optimized on external control tissues, which were included as batch controls. Dye swaps ensured specificity of double staining.

研究目的：Fuchs’内皮角膜营养不良（Fuchs’ endothelial corneal dystrophy, FECD）是一种退行性眼部疾病，影响4%的40岁以上美国人群，亦是角膜内皮（corneal endothelial, CE）移植的首要适应证，而全球范围内存在角膜供体短缺的问题。本研究旨在进一步阐明FECD的病理生理学机制，并筛选非手术治疗的潜在靶点。
研究方法：本研究采用微阵列表达分析（microarray expression analysis，FECD组n=4，正常对照组n=4）、逆转录定量PCR（reverse transcriptase quantitative PCR, RT-qPCR，FECD组n=9，正常对照组n=8）以及免疫组织化学检测（FECD组n=55，正常对照组n=15），对比晚发性FECD患者的角膜内皮组织与正常对照组织的差异。
研究结果：本研究在所有受检的FECD患者角膜内皮样本中（涵盖有症状FECD的所有临床分期，且与既往白内障手术史无关），均检测到循环纤维细胞及其树突状衍生物；而正常角膜内皮组织中未发现此类细胞。本研究对这类细胞的形态学特征、蛋白表达谱、数量以及在FECD患者角膜内皮层中的定位进行了系统表征。
研究结论：循环纤维细胞及其树突状衍生物是FECD发病机制中一个全新的未被报道的环节，值得进一步深入研究。鉴于此类细胞已被证实可在多种器官中诱导纤维化，它们可能在FECD进程中发挥类似作用，或可成为非手术治疗的潜在有效靶点。
总体实验设计：本研究的实验材料为携带后弹力膜（Descemet membrane）的晚发性FECD患者角膜内皮单层细胞。微阵列表达分析共纳入8例样本（FECD组4例，正常对照组4例）。针对179个目的基因及至少3个内参基因开展RT-qPCR检测（FECD组n=9，正常对照组n=8）。通过免疫组织化学与免疫荧光染色，对新鲜角膜内皮整体铺片（whole-mount）中的目的分子进行验证（FECD组n=55，正常对照组n=15）。所有染色均以外部对照组织进行优化，并将其作为批次对照纳入实验。采用染料互换（dye swaps）技术确保双重染色的特异性。