Department of Ophthalmology, Duke University Eye Center, Durham, North Carolina 27713, USA.
Invest Ophthalmol Vis Sci. 2010 Dec;51(12):6483-95. doi: 10.1167/iovs.10-5410. Epub 2010 Jun 23.
To investigate the role of intralysosomal redox-active iron in oxidative stress-induced damage in trabecular meshwork (TM) cells.
Chronic oxidative stress was applied using the hyperoxic model; acute oxidative stress was applied with H(2)O(2). Microarray analysis was performed using microarrays. mRNA and protein levels were quantified by real-time PCR and Western blot analysis, respectively. Redox-active iron was monitored using calcein-AM. Apoptosis was quantified using double staining. DNA damage was evaluated by single-cell gel electrophoresis assay. Lysosomal permeabilization was monitored using uptake and acridine orange relocation techniques. Intracellular ROS production was quantified using H(2)DCFDA. Cytosolic translocation of cathepsins was visualized with pepstatin-A-BODIPY-FL. Chemical inhibition of cathepsins was achieved with leupeptin and pepstatin A. Silencing of cathepsin expression was accomplished with miRNA sequences. Lysosomal iron chelation was achieved with desferrioxamine.
Chronically stressed TM cells showed elevated levels of redox-active iron and altered expression of genes involved in intracellular iron homeostasis. Although iron increased ROS production and lipofuscin levels and sensitized TM cells to H(2)O(2), intralysosomal iron chelation completely protected the cells against H(2)O(2)-induced cell death and apoptosis. The protective effect of desferrioxamine was mediated by the prevention of lysosomal ROS generation and the rupture of lysosomal membrane, with the subsequent release of cathepsin D into the cytosol.
These results indicate that the generation of intralysosomal ROS induces lysosomal membrane permeabilization and the release of cathepsin D into the cytosol, leading to TM cell death. Here, the authors propose a mechanism by which oxidative stress might contribute to the decrease in cellularity reported in the TM tissue with both aging and disease.
研究细胞内溶酶体中具有氧化还原活性的铁在小梁网（TM）细胞氧化应激损伤中的作用。
通过高氧模型应用慢性氧化应激；通过 H2O2 应用急性氧化应激。使用微阵列进行微阵列分析。通过实时 PCR 和 Western blot 分析分别定量 mRNA 和蛋白质水平。使用 calcein-AM 监测具有氧化还原活性的铁。通过双染色定量细胞凋亡。通过单细胞凝胶电泳试验评估 DNA 损伤。通过摄取和吖啶橙重定位技术监测溶酶体通透性。通过 H2DCFDA 量化细胞内 ROS 产生。用 pepstatin-A-BODIPY-FL 可视化组织蛋白酶的细胞质易位。用亮抑酶肽和胃蛋白酶抑制剂 A 实现组织蛋白酶的化学抑制。用 miRNA 序列实现组织蛋白酶表达的沉默。用去铁胺实现溶酶体铁螯合。
慢性应激 TM 细胞显示出氧化还原活性铁水平升高和参与细胞内铁稳态的基因表达改变。虽然铁增加了 ROS 产生和脂褐素水平，并使 TM 细胞对 H2O2 敏感，但细胞内溶酶体铁螯合完全防止了 H2O2 诱导的细胞死亡和凋亡。去铁胺的保护作用是通过防止溶酶体 ROS 产生和溶酶体膜破裂，随后将组织蛋白酶 D 释放到细胞质中来介导的。
这些结果表明，细胞内 ROS 的产生诱导溶酶体膜通透性和组织蛋白酶 D 释放到细胞质中，导致 TM 细胞死亡。在这里，作者提出了一种机制，即氧化应激可能导致 TM 组织中细胞数量减少，这与衰老和疾病都有关。
