Chandler Heather L, Gervais Kristen J, Lutz Elizabeth A, Curto Elizabeth M, Matusow Rachel B, Wilkie David A, Gemensky-Metzler Anne J
The Ohio State University, College of Optometry, 320 West 10th Avenue, Columbus, OH 43210, USA; The Ohio State University, College of Veterinary Medicine, 601 Vernon Tharp Street, Columbus, OH 43210, USA.
The Ohio State University, College of Optometry, 320 West 10th Avenue, Columbus, OH 43210, USA.
Exp Eye Res. 2015 May;134:63-72. doi: 10.1016/j.exer.2015.03.020. Epub 2015 Mar 31.
The purpose of this study was to determine the Cyclosporine A (CsA) dose and minimum drug delivery time needed to prevent posterior capsule opacification (PCO) in an ex vivo canine model and evaluate the mechanism of CsA-induced cell death. Canine lens epithelial cells (LEC) were treated with CsA and changes in cell migration, proliferation, and density were monitored over time. CsA-treated LEC underwent transmission electron microscopy (TEM), immunofluorescence, and immunoblotting in the presence or absence of autophagy inhibitors to evaluate the mechanism of cell death. Lens capsules were harvested from canine cadaver eyes for an ex vivo model of PCO. Lens capsules were treated with CsA for 1, 2, 3, 4, 5, 6, or 7 days, and subsequently maintained in culture for a total of 28 days in the absence of drug. CsA reduced LEC viability in a dose dependent manner. Morphologically, CsA-treated LEC were swollen, had intact nuclei, lacked peripheral chromatin condensation, and demonstrated prominent vacuolization; TEM revealed autophagosomes. LC3-II protein expression and acridine orange fluorescence increased in CsA-treated cells. A small non-significant induction of cleaved caspase-3 was observed in CsA-treated LEC. Lens capsules treated with 5, 6, or 7 days of 10 μg/mL CsA showed a significant decrease in ex vivo PCO formation; 6 days of drug delivery prevented PCO. This study finds that morphologic changes, formation of acidic vesicles, and increased expression of LC3-II supports the hypothesis that CsA mediates LEC death via autophagy; this is a novel finding in the lens. Induction of CsA-induced apoptosis was minimal. Six days of intracapsular CsA drug delivery prevented ex vivo PCO formation.
本研究的目的是确定环孢素A（CsA）的剂量和预防体外犬模型后囊膜混浊（PCO）所需的最短药物递送时间，并评估CsA诱导细胞死亡的机制。用CsA处理犬晶状体上皮细胞（LEC），并随时间监测细胞迁移、增殖和密度的变化。在有或没有自噬抑制剂的情况下，对经CsA处理的LEC进行透射电子显微镜（TEM）、免疫荧光和免疫印迹分析，以评估细胞死亡机制。从犬尸体眼睛中获取晶状体囊膜用于PCO的体外模型。将晶状体囊膜用CsA处理1、2、3、4、5、6或7天，随后在无药物的情况下总共培养28天。CsA以剂量依赖性方式降低LEC活力。形态学上，经CsA处理的LEC肿胀，细胞核完整，缺乏外周染色质凝聚，并表现出明显的空泡化；TEM显示有自噬体。经CsA处理的细胞中LC3-II蛋白表达和吖啶橙荧光增加。在经CsA处理的LEC中观察到少量无统计学意义的裂解型半胱天冬酶-3的诱导。用10μg/mL CsA处理5、6或7天的晶状体囊膜在体外PCO形成方面显著降低；6天的药物递送可预防PCO。本研究发现，形态学变化、酸性囊泡的形成以及LC3-II表达的增加支持了CsA通过自噬介导LEC死亡的假说；这在晶状体中是一个新发现。CsA诱导的凋亡极少。囊内CsA药物递送6天可预防体外PCO形成。
