Abstract
Purpose: :
To investigate the effects of chronic oxidative stress on lysosomal function in the cells of the outflow pathway.
Methods: :
Confluent cultures of porcine trabecular meshwork (TM) cells were grown for two weeks at physiological (5% O2) or hyperoxic conditions (40% O2) in the presence or absence of the lysosomal inhibitor leupeptin (10 µM). The following parameters were quantified by flow cytometry using the fluorogenic probes indicated within parentheses: autofluorescence, intracellular ROS (H2DCFDA), mitochondrial membrane potential (JC-1), mitochondrial content (mitotracker red), lysosomal content (acridine orange and lysotracker red), autophagic vacuoles content (MDC), and SA-β-galactosidase (FDG). Cathepsin levels were quantified by real-time PCR and Western-blot analysis. Cathepsin activities were evaluated using the omnicathepsins fluorogenic substrate z-FR-AMC. Ultrastructural analysis was performed by transmission electron microscopy.
Results: :
Chronic exposure of porcine TM cells to a hyperoxic environment led to a statistically significant increase in ROS production (104.42±50.85%, p=0.026) and oxidized material (156.24 ± 85.85%, p=0.035). Electron micrographs revealed the cytoplasmic accumulation of autophagic secondary lysosomes containing electron dense material and membranous structures in the perinuclear region of the stressed cells. Augmented lysosomal and autophagic vacuoles content was confirmed using specific fluorophores (323.9±88.30%, p=0.005 and 51.29±33.05, p=0.01, respectively). The stressed cultures also demonstrated increased mitochondrial mass (96.94±21.69%, p=0.008) accompanied by decreased mitochondrial membrane potential (27.86±4.11%, p=0.007), suggesting the accumulation of defective mitochondria following oxidative stress. The mRNA and protein levels of several cathepsins were found to be upregulated with oxidative stress. However, this upregulated expression did not correlate with increased lysosomal activity.
Conclusions: :
Our results indicate that chronic exposure of TM cells to oxidative stress induces the accumulation of nondegradable material within the lysosomal compartment leading to diminished lysosomal activity. Since the lysosomal system is responsible for the continuous turnover of cellular organelles (specially mitochondria) and processing of exogenous phagocytosed material, diminished lysosomal activity may lead to progressive failure of the cellular TM function with age, and thus possibly contribute to the pathogenesis of primary open angle glaucoma.
Keywords: trabecular meshwork • aging • oxidation/oxidative or free radical damage