Intralysosomal iron induces lysosomal membrane permeabilization and cathepsin D-mediated cell death in trabecular meshwork cells exposed to oxidative stress.

Aging of the trabecular meshwork (TM), the tissue responsible for maintaining proper levels of intraocular pressure (IOP), is suspected to be a major risk factor for the development or progression of primary open-angle glaucoma (POAG), a late-onset disease that constitutes the second leading cause of permanent blindness worldwide. Evidence suggests that an acceleration in the production of reactive oxygen species (ROS) causes oxidative damage to the TM with aging1–5 and that this might contribute to the observed loss in TM tissue functionality in ocular hypertension and in POAG.6–8 However, the specific mechanisms by which free radicals may exert these pathologic effects on the outflow pathway and the true source of ROS have not yet been determined or even fully hypothesized. A potential source of intracellular ROS is that generated from H2O2 through iron-catalyzed Fenton reactions, which result in the production of highly reactive hydroxyl radicals (H2O2 + Fe2+ → Fe3+ + OH + OH−). These newly generated free radicals can induce the peroxidation of adjacent lipids and proteins and oxidative damage to DNA.9 To meet the physiological demands while avoiding the pro-oxidant effect of iron, cells and organisms handle iron with extreme caution and efficiently prevent free iron from reacting with H2O2 by sequestering it within a series of proteins that control either iron uptake (transferrin; transferrin receptor [TFRC]), iron storage (ferritin, composed of ferritin heavy chain [FTH]; and ferritin light chain [FTL]), or iron export (ferroportin [SLC-40]; ceruloplasmin [CP]). Moreover, transferrin-mediated iron uptake and iron storage in ferritin are events controlled by the concentration of free redox-active iron (labile iron pool [LIP]) through the coordinated action of cytosolic iron regulatory proteins (IRPs), which bind to iron-responsive element (IRE) motifs present in the untranslated regions of TFRC and FTH/FTL genes. Thus, a reduction in intracellular iron availability causes IRPs to bind the IRE motifs in the mRNAs of TFRC or FTH/FTL, thereby inhibiting the translation of ferritin and stabilizing the TFRC mRNA to favor iron uptake and decrease storage. In contrast, under high iron concentrations, IRPs are either assembled into clusters or degraded by the proteolytic systems and cannot bind IRE motifs. This situation results in an upregulation of ferritin synthesis and an increase of TFRC mRNA degradation, thus promoting iron storage and inhibiting iron uptake.10–12 Intracellular iron levels have been reported to increase exponentially in cultured primary human fibroblasts and in umbilical vein endothelial cells as a function of cellular senescence.13,14 Similarly, localized iron dysregulation with consequent iron accumulation has been demonstrated to occur in a variety of tissues and species during normal aging.15–22 It has been postulated that such deposition of iron might contribute to the increased oxidative stress and cellular dysfunction associated with normal aging and with the pathology of a growing number of age-related diseases, including Alzheimer's disease, Parkinson's disease, type 2 diabetes, cardiovascular diseases, and macular degeneration.16,20,22–25 Although the mechanisms by which iron accumulates intracellularly during aging are still largely unknown, lysosomes have been suggested to play a major role in the intracellular regulation and homeostasis of iron metabolism.26–28 Lysosomes are the main cellular organelles responsible for the degradation of organelles and long-lived proteins and of extracellular and membrane-bound material. Because of the breakdown of iron-containing endocytosed and autophagocytosed material, significant concentrations of labile iron accumulate within the lysosomes,15,29 where it participates in Fenton reactions, leading to the generation of lysosomal ROS. Our laboratory recently reported that prolonged exposure of TM primary cultures to a hyperoxic environment, as an in vitro model of aging, led to a striking increase in ROS production. This increase in iROS production was accompanied by alterations in the lysosomal pathway in the stressed cultures.30 The purposes of this study were to evaluate the effect of oxidative stress in intracellular iron homeostasis in TM cells and the potential role of intralysosomal iron in outflow pathway pathophysiology. The data presented here provide evidence for an accumulation of redox active iron in TM cells exposed to chronic oxidative stress. Furthermore, we propose a mechanism by which iron-catalyzed oxidative damage might contribute to the decrease in cellularity reported in the TM tissue with both aging and disease.